Mammalian T1R3 sweet taste receptors

ABSTRACT

The present invention provides isolated nucleic acid and amino acid sequences of sweet taste receptors, the receptors comprising consisting of a monomer or homodimer of a T1R3 G-protein coupled receptor polypeptide, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of sweet and amino acid taste receptors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.10/679,102, filed Oct. 2, 2003, which is herein incorporated byreference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention provides isolated nucleic acid and amino acidsequences of sweet taste receptors, the receptors comprising consistingof a monomer or homodimer of a T1R3 G-protein coupled receptorpolypeptide, antibodies to such receptors, methods of detecting suchnucleic acids and receptors, and methods of screening for modulators ofsweet taste receptors.

BACKGROUND OF THE INVENTION

The sense of taste is responsible for detecting and responding to sweet,bitter, sour, salty and umami (amino acid) stimuli. It is also capableof distinguishing between these various taste modalities to generateinnate behavioral responses. For instance, animals are vigorously averseto bitter-tasting compounds, but are attracted to sweet and umamistimuli. To examine taste signal detection and information processing,we have focused on the isolation and characterization of sweet, umamiand bitter taste receptors. These receptors provide powerful moleculartools to delineate the organization of the taste system, and to helpdefine the logic of taste coding.

Two families of candidate mammalian taste receptors, the T1Rs and T2Rs,have been implicated in sweet, umami and bitter detection. The T2Rs area family of ˜30 taste-specific GPCRs distantly related to opsins, andclustered in regions of the genome genetically linked to bitter taste inhumans and mice (Adler et al., Cell 100, 693-702 (2000); Matsunami etal., Nature, 404, 601-604 (2000)). Several T2Rs have been shown tofunction as bitter taste receptors in heterologous expression assays,substantiating their role as bitter sensors (Chandrashekar et al., Cell,100, 703-711 (2000); Bufe et al., Nat Genet, 32, 397-401 (2002)). MostT2Rs are co-expressed in the same subset of taste receptor cells (Adler,E. et al., Cell 100, 693-702 (2000)), suggesting that these cellsfunction as generalized bitter detectors.

The T1Rs are a small family of 3 GPCRs expressed in taste cells of thetongue and palate epithelium, distantly related to metabotropicglutamate receptors, the calcium sensing receptor and vomeronasalreceptors (Hoon et al., Cell., 96, 541-551 (1999); Kitagawa et al.,Biochem Biophys Res Commun, 283, 236-242 (2001); Max et al.,| Sac. NatGenet, 28, 58-63 (2001); Montmayeur et al. Nat Neurosci, 4, 492-498(2001); Nelson et al., Cell, 106, 381-390 (2001); Sainz et al., JNeurochem, 77, 896-903 (2001)). T1Rs combine to generate at least twoheteromeric receptors: T1R1 and T1R3 form an L-amino acid sensor, whichin rodents recognizes most amino acids, and T1R2 and T1R3 associate tofunction as a broadly tuned sweet receptor (Nelson, G. et al., Cell,106, 381-390 (2001); Nelson, G. et al., Nature, 416, 199-202 (2002); Li,X. et al., Proc Natl Acad Sci USA, 99, 4692-4696 (2002); see also WO00/06592, WO 00/06593, and WO 03/004992).

Animals can detect a wide range of chemically distinct sweet tastingmolecules, including natural sugars, artificial sweeteners, D-aminoacids and intensely sweet proteins. How many different receptors does ittake to taste the sweet universe? The human and rodent T1R2+3heteromeric sweet receptors respond in cell-based assays to all classesof sweet compounds, and do so with affinities that approximate theirrespective in vivo psychophysical and/or behavioral thresholds (Nelsonet al., Cell, 106, 381-390 (2001); Li et al., Proc Natl Acad Sci USA,99, 4692-4696 (2002)). At a fundamental level, the evolution of sweettaste most likely reflects the need to detect and measure sugar contentin potential food sources. Therefore, a single broadly tuned receptorfor natural sugars might be all that is required. On the other hand, anumber of studies with various sugars and artificial sweetenersinsinuate the possibility of more than one sweet taste receptor(Schiffman et al., Pharmacol Biochem Behav, 15, 377-388 (1981); Ninomiyaet al., J Neurophysiol, 81, 3087-3091 (1999)).

In humans, monosodium L-glutamate (MSG) and L-aspartate, but not otheramino acids, elicit a distinctive savory taste sensation called umami(Maga, 1983). Notably, unlike the rodent T1R1+3, the human T1R1+3 aminoacid taste receptor is substantially more sensitive to L-glutamate andL-aspartate than to other L-amino acids (Li et al., Proc Natl Acad SciUSA, 99, 4692-4696 (2002)). These findings led to the proposal thatT1R1+3 may be the mammalian umami receptor (Nelson. et al., Nature, 416,199-202 (2002); Li. et al., Proc Natl Acad Sci USA, 99, 4692-4696(2002)). However, a number of studies, including the recent analysis ofT1R3 KO mice (Damak et al., Science, 301, 850-853 (2003)) have suggestedthat umami taste is mediated by mGluR4t, a truncated variant of themetabotropic glutamate receptor (Chaudhari et al., Neurosci, 16,3817-3826 (1996); Chaudhari. et al., Nat Neurosci, 3, 113-119 (2000)).

How are the different taste qualities encoded at the taste cell level?In mammals, taste receptor cells are assembled into taste buds that aredistributed in different papillae in the tongue epithelium. Each tastebud contains 50-150 cells, including precursor cells, support cells, andtaste receptor cells (Lindemann, Physiol Rev, 76, 718-766 (1996)). Thereceptor cells are innervated by afferent fibers that transmitinformation to the taste centers of the cortex through synapses in thebrain stem and thalamus. In the simplest model of taste coding at theperiphery, each taste modality would be encoded by a unique populationof cells expressing specific receptors (e.g. sweet cells, bitter cells,salt-sensing cells, etc.). In this scenario, our perception of any onetaste quality would result from the activation of distinct cell types inthe tongue (labeled line model). Alternatively, individual taste cellscould recognize multiple taste modalities, and the ensemble firingpattern of many such broadly tuned receptor cells would encode tastequality (across fiber model).

Recently, we showed that T1Rs and T2Rs are expressed in completelynon-overlapping populations of receptor cells in the lingual epithelium(Nelson et al., Cell, 106, 381-390 (2001)), and demonstrated thatbitter-receptor expressing cells mediate responses to bitter but not tosweet or amino acid tastants (Zhang et al., Cell, 112, 293-301 (2003)).Together, these results argued that taste receptor cells are not broadlytuned across all modalities, and strongly supported a labeled line modelof taste coding at the periphery. A fundamental question we address nowis how many types of cells and receptors are necessary to mediate sweetand umami, the two principal attractive taste modalities. We now showthat sweet and umami tastes are exclusively mediated by T1Rs, anddemonstrate that genetic ablation of individual T1R subunits selectivelyaffects these two attractive taste modalities. The identification ofcells and receptors for sweet and umami sensing also allowed us todevise a strategy to separate the role of receptor activation from cellstimulation in encoding taste responses. We show that animals engineeredto express a modified k-opioid receptor in T1R2+3-expressing cellsbecome specifically attracted to a k-opioid agonist, and prove thatactivation of sweet-receptor expressing cells, rather than the T1Rreceptors themselves, is the key determinant of behavioral attraction tosweet tastants. Finally, we now demonstrate that T1R1 alone, either as amonomer or as a homodimer, acts as a receptor for naturally occurringsugars.

BRIEF SUMMARY OF THE INVENTION

The present invention thus provides for the first time a homodimericsweet taste receptor, the receptor comprising or consisting of two T1R3polypeptides. The present invention also provides a monomeric sweettaste receptor comprising or consisting of one T1R3 polypeptide. Thereceptors transduce a signal in response to sweet taste ligands whenT1R3 is expressed in a cell. In one embodiment, the sweet taste ligandsare naturally occurring sweet tasting molecules. In another embodiment,the sweet taste ligands and artificial and mimic naturally occurringsweet tasting molecules. In one embodiment, the T1R3 polypeptides of thehomodimer are non-covalently linked.

In one aspect, the present invention provides a sweet taste receptorcomprising a T1R3 polypeptide, the T1R3 polypeptide comprising greaterthan about 80% amino acid sequence identity to an amino acid sequence ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31or encoded by a nucleotide sequence hybridizing under moderately orhighly stringent hybridization conditions to a nucleotide sequenceencoding an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:25, or SEQ ID NO:31.

In one embodiment, the T1R3-comprising receptor specifically binds topolyclonal antibodies generated against SEQ ID NO:15, SEQ ID NO:20, SEQID NO:23, SEQ ID NO:25, or SEQ ID NO:31. In another embodiment, thereceptor has G-protein coupled receptor activity. In another embodiment,the T1R3 polypeptide has an amino acid sequence of SEQ ID NO:15, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31. In anotherembodiment, the receptor is from a human, a rat, or a mouse.

In another embodiment, the sweet receptor comprises a T1R3 polypeptideand recognizes natural sugars, e.g., glucose, galactose, fructose,maltose, lactose, and sucrose.

In one aspect, the present invention provides an isolated polypeptidecomprising an extracellular, a transmembrane domain, or a cytoplasmicdomain of a sweet T1R3-comprising homodimeric or monomeric tastereceptor, the extracellular, a transmembrane domain, or a cytoplasmicdomain comprising greater than about 80% amino acid sequence identity tothe extracellular, a transmembrane domain, or a cytoplasmic domain ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.In another embodiment, the extracellular, transmembrane, or cytoplasmicdomain hybridize under highly stringent conditions to an extracellular,transmembrane, or cytoplasmic domain of an amino acid sequence of SEQ IDNO:15, 20, 23, 25, or 31.

In one embodiment, the polypeptide encodes the extracellular, atransmembrane domain, or a cytoplasmic domain of SEQ ID NO:15, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31. In anotherembodiment, the extracellular, a transmembrane domain, or a cytoplasmicdomain is covalently linked to a heterologous polypeptide, forming achimeric polypeptide. In another embodiment, the chimeric polypeptidehas G-protein coupled receptor activity.

In one aspect, the present invention provides an antibody thatselectively binds to a homodimeric or monomeric sweet taste receptor,the receptor comprising one or two T1R3 polypeptides but no T1R1 or T1R2polypeptides, the antibody raised against a receptor comprising a T1R3polypeptide comprising greater than about 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:31 or encoded by a nucleotide sequencehybridizing under highly stringent hybridization conditions to anucleotide sequence encoding an amino acid sequence of SEQ ID NO:15, SEQID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.

In another aspect, the present invention provides a method foridentifying a compound that modulates sweet taste signaling in tastecells, the method comprising the steps of: (i) contacting the compoundwith a homodimeric or monomeric receptor comprising a T1R3 polypeptidebut not a T1R1 or a T1R2 polypeptide, the polypeptide comprising greaterthan about 80% amino acid sequence identity to SEQ ID NO: 15, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31; or encoded by anucleotide sequence hybridizing under highly stringent hybridizationconditions to a nucleotide sequence encoding an amino acid sequence ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31;and (ii) determining the functional effect of the compound upon thereceptor.

In one embodiment, the functional effect is determined in vitro. In oneembodiment, the polypeptide is expressed in a cell or cell membrane. Inanother embodiment, the receptor is linked to a solid phase, eithercovalently or non-covalently.

In another aspect, the present invention provides a method foridentifying a compound that modulates sweet taste signaling in tastecells, the method comprising the steps of: (i) contacting a cell withthe compound, the cell expressing a homodimeric or monomeric receptorcomprising a T1R3 polypeptide but not expressing a T1R1 or a T1R2polypeptide, the T1R3 polypeptide comprising greater than about 80%amino acid sequence identity to SEQ ID NO:15, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:25, or SEQ ID NO:31; or encoded by a nucleotidesequence hybridizing under highly stringent hybridization conditions toa nucleotide sequence encoding an amino acid sequence of SEQ ID NO:15,SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31; and (ii)determining the functional effect of the compound upon the receptor.

In one embodiment, the functional effect is determined by measuringchanges in intracellular cAMP, IP3, or Ca2+. In another embodiment, thefunctional effect is a chemical or phenotypic effect. In anotherembodiment, the functional effect is a physical effect. In anotherembodiment, the functional effect is determined by measuring binding ofthe compound to the extracellular domain of the receptor. In anotherembodiment, the polypeptide is recombinant. In another embodiment, thecell is a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell.In another embodiment, the cell expresses G protein Gα15.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Targeted KO of T1R1, T1R2 and T1R3.

(a) Schematic drawing showing the structure of the three T1R genes andthe strategy for generating knockout animals. The targeting constructsdeleted all seven predicted transmembrane helices of T1R1 and T1R2, andthe entire extracellular ligand binding domain of T1R3. (b) In situhybridization labeling demonstrating robust expression of T1Rs in tastebuds of wild-type animals, but complete absence in the correspondingknock-out mice.

FIG. 2: T1R mutants respond normally to sour, salty and bitter stimuli

(a) Wild-type (WT), T1R1, T1R2 and T1R3 knockout mice (1-KO, 2-KO, 3-KO)show robust neural responses to sour (100 mM citric acid), salty (100 mMNaCl) and bitter (10 mM PROP) tastants. (b) Integrated neural responses,such as those shown in (a), were normalized to the response elicited by100 mM citric acid; control and KO animals are indistinguishable fromeach other. The values are means ± s.e.m. (n=4). The data representchorda tympani responses (see Experimental Procedures for details). (c),Taste preferences of wild-type and T1R knockout animals were measuredrelative to water using a brief access taste test (Zhang, Y. et al.,Cell, 112, 293-301 (2003)). All four lines showed normal responses tosour, salty and bitter stimuli. The values are means± s.e.m. (n=7).Similar results were obtained using a standard two bottle preferenceassay (data not shown). Cyx, cycloheximide; Den, denatonium benzoate;PROP, 6-n-propyl-thiouracil; Qui, quinine.

FIG. 3: T1R1+3 functions as the mammalian umami receptor

(a-d) Taste preferences of wild-type (open circles, dashed lines), T1R1KO (blue circles and bars), T1R2 KO (gray circles and bars) and T1R3 KOmice (brown circles and bars) were measured relative to water using abrief access taste test. T1R2 KO mice are equivalent to wild typecontrols. In contrast, T1R1 and T1R3 knockout animals exhibit a completeloss in preference for umami tastants (a) MSG+1 mM IMP, (b) MSG, (c)IMP, and (d) L-Asp (100 mM), and AP4 (30 mM). In addition, both knockouthave marked impairments in other amino acid responses. L-Asn (100 mM)and L-Arg were used at 100 mM each. (e-f) Integrated chorda tympaniresponses to umami tastants and amino acids. T1R1 and T1R3 knockoutshave a complete loss of responses to (e) umami agonists and L-aminoacids if salt effects are avoided by using either amiloride or thepotassium salt of MSG (MPG). In contrast, (f) if high concentrations ofsalt are used (e.g. 100 mM MSG), residual responses are detected.

FIG. 4: T1R2 and T1R3 are essential for sweet taste perception

(a) Taste preferences of wild-type (open circles, dashed lines), T1R1 KO(gray circles and bars), T1R2 KO (green circles and bars) and T1R3 KOmice (brown circles and bars) were measured relative to water using abrief access taste test. T1R1 KO mice are equivalent to wild typecontrols. In contrast, T1R2 and T1R3 knockout animals exhibit a completeloss in preference for artificial sweeteners and D-amino acids, butretain residual responses to high concentration of natural sugars. Theseare highlighted in (b) as dose responses in expanded scale for maltose,sucrose and glucose. However, T1R2/T1R3 double KO animals (red circles)have a complete loss of all sweet responses. The values are Means ±s.e.m. (n=7). D-Asn and D-Phe were 100 mM each, and D-Trp was used at 30mM.

FIG. 5: T1R2 and T1R3 encode the mammalian sweet taste receptors

Panel (a) shows integrated chorda tympani responses to natural sugars,artificial sweeteners and D-amino acids in wild type (WT) and T1Rknockout animals (1-KO, 2-KO, 3-KO). T1R2 and T1R3 knockouts have acomplete loss of responses to artificial sweeteners and D-amino acid(red traces), but show small neural responses to high concentrations ofnatural sugars. These, however, are completely abolished in T1R2/T1R3double KO mice (bottom red traces). Panel (b) shows average neuralresponses to an expanded panel of tastants; wild type, white bars; T1R2KO, green bars; T1R3 KO, brown bars; T1R2/T1R3 double KO, red bars. Thevalues are means ± s.e.m. (n=4) of normalized chorda tympani responses.

FIG. 6. T1R3 responds to high concentrations of natural sugars

HEK-293 cells co-expressing the promiscuous G protein G_(gust-25) (seeExperimental Procedures) and the mouse T1R3 GPCR, or co-transfected withboth T1R2 plus T1R3, were stimulated with various sweet compounds. Upperpanels show increases in [Ca²⁺]i upon stimulation of T1R3-expressingcells with 500 mM, but not 300 mM sucrose. No responses were detectedwith artificial sweeteners (300 mM saccharin, right panel), or in cellswithout receptors or G_(gust-25); scale indicates [Ca²⁺]i (nM)determined from FURA-2 F₃₄₀/F₃₈₀ ratios. As expected, control cellsexpressing T1R2+3 (lower panels) respond robustly to lowerconcentrations of natural (300 mM sucrose) and artificial sweeteners (30mM saccharin).

FIG. 7. Activation of T1R2-expressing cells triggers behavioralattraction

(a) Wild type and T1R2 KO mice expressing a human T1R2 gene under thecontrol of the rodent T1R2-promoter were (b-d) tested for behavioralresponses to a variety of human sweet tastants: (b) Ace-K, acesulfame-K,(c) aspartame, and (d) MON, monellin (˜10 μM); THAU, thaumatin (˜5 μM);ASP, aspartame (10 mM); GA, glycyrrhizic acid (500 μM); NH,neohesperidin dihydrochalcone (400 μM). The human T1R2 taste receptor is(a) selectively expressed in T1R2-cells, and (b) effectively rescuessweet taste responses of T1R2 KO mice. Importantly, the presence of thetransgene (c-d) humanizes the sweet taste preferences of the transgenicanimals. See text for details. (e) Expression of RASSL (Redfern, C. H.et al., Nat Biotechnol, 17, 165-169 (1999)) in T1R2-cells generatesanimals that exhibit specific behavioral attraction to spiradoline. Notethat no responses are seen in uninduced animals, or control mice, evenat 100× the concentration needed to elicit strong responses inRASSL-expressing animals. The values are means ± s.e.m. (n=7)

FIG. 8

FIG. 8 provides a nucleotide sequence of hT1R1 (SEQ ID NO:26).

FIG. 9

FIG. 9 provides an amino acid sequence of hT1R1 (SEQ ID NO:27).

FIG. 10

FIG. 10 provides a nucleotide sequence of hT1R2 (SEQ ID NO:28).

FIG. 11

FIG. 11 provides a amino acid sequence of hT1R2 (SEQ ID NO:29).

FIG. 12

FIG. 12 provides a nucleotide sequence of hT1R3 (SEQ ID NO:30).

FIG. 13

FIG. 13 provides an amino acid sequence of hT1R3 (SEQ ID NO:31).

DETAILED DESCRIPTION OF THE INVENTION INTRODUCTION

T1Rs and T2Rs are two families of G-protein-coupled receptors (GPCRs)selectively expressed in subsets of taste receptor cells (Hoon et al.,Cell 96:541-551 (1999); Adler et al., Cell 100:693-702 (2000);Chandrashekar et al., Cell 100:703-711 (2000); Matsunami et al., Nature404:601-604 (2000); Nelson et al., Cell 106:381-390 (2001); Kitagawa etal., Biochem. Biophys. Res. Cummun. 283:236-242 (2001); Montmayeur etal., Nature Neurosci. 4:492-498 (2001); Max et al., Nature Genet.28:58-63 (2001); Sainz et al., J. Neurochem. 77:896-903 (2001)). T2Rsare involved in bitter taste detection (Adler et al., Cell 100:693-702(2000); Chandrashekar et al. Cell, 100:703-711 (2000)); T1R2 and T1R3combine to function as a sweet taste receptor (see also Nelson et al.,Cell 106:381-390 (2001); and T1R1 and T1R3 combine to function as anamino acid taste receptors, as described herein (see also Nelson et al.,Nature 24 Feb. 2002 and WO 03/004992))). We have now identified ahomodimeric taste receptor, in which two T1R3 polypeptides combine tofunction as a sweet taste receptor. The monomeric form of T1R3 also actsas a sweet receptor.

Using a heterologous expression system, we demonstrate that T1R3combines with itself and also acts as a monomer to function as a sweetreceptor, recognizing sweet-tasting molecules such as sucrose,galactose, fructose, glucose, maltose, and lactose. Candidate receptorsare expressed in human embryonic kidney (HEK) cells containing theGα₁₆-Gα_(z) and Gα₁₅ promiscuous G proteins (Offermanns et al., J. Biol.Chem. 270:15175-15180 (1995); Mody et al., Mol. Pharmacol. 57:13-23(2000)), and assayed for stimulus-evoked changes in intracellularcalcium. In this system, receptor activation leads to activation ofphospholipase Cβ (PLC-β and release of calcium from internal stores,which can be monitored at the single-cell level using calcium-indicatordyes (Chandrashekar et al., Cell 100:703-711 (2000); Nelson et al., Cell106:381-390 (2001); Tsien et al., Cell Calcium 6:145-157 (1985)).

These nucleic acids and proteins encoding the receptors provide valuableprobes for the identification of taste cells, as the nucleic acids arespecifically expressed in taste cells. The receptors are useful forassaying for novel tastants, such as artificial sweetener molecules. Forexample, probes for GPCR polypeptides and proteins can be used toidentity subsets of taste cells such as foliate cells, palate cells, andcircumvallate cells, or specific taste receptor cells, e.g., sweet tastereceptor cells. They also serve as tools for the generation of tastetopographic maps that elucidate the relationship between the taste cellsof the tongue and taste sensory neurons leading to taste centers in thebrain. Furthermore, the nucleic acids and the proteins they encode canbe used as probes to dissect taste-induced behaviors.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel monomeric or homodimeric sweet tastereceptors comprising T1R3. In one embodiment, the monomeric orhomodimeric T1R3-comprising receptors of the invention can be used toscreen for naturally occurring or artificial sweet tasting molecules ormodulators of sweet taste transduction, e.g., small organic molecules,amino acids, peptides, carbohydrates, lipids, polysaccharides, etc. Forexample, homodimeric or monomeric T1R3-comprising receptors of theinvention recognize naturally occurring sweet tastants, as describedbelow in the example section. Such receptors can be used to screen forartificial sweeteners, or altered naturally occurring sweeteners, thatmimic the naturally occurring sugar ligands of the homodimeric ormonomeric T1R3-comprising receptor. Such modulators of sweet tastetransduction are useful for pharmacological and genetic modulation ofsweet taste signaling pathways, and for the discovery of novel sweettaste ligands. These methods of screening can be used to identifyagonists and antagonists of sweet taste cell activity. These modulatorycompounds can then be used in the food and pharmaceutical industries tocustomize taste. Thus, the invention provides assays for tastemodulation, where the T1R3-comprising receptor acts as an direct orindirect reporter molecule for the effect of modulators on sweet tastetransduction. GPCRs can be used in assays, e.g., to measure changes inligand binding, G-protein binding, regulatory molecule binding, ionconcentration, membrane potential, current flow, ion flux,transcription, signal transduction, receptor-ligand interactions,neurotransmitter and hormone release; and second messengerconcentrations, in vitro, in vivo, and ex vivo. In one embodiment, areceptor comprising T1R3 can be used as an indirect reporter viaattachment to a second reporter molecule such as green fluorescentprotein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964(1997)). In another embodiment, a receptor comprising T1R3 isrecombinantly expressed in cells that do not express either T1R1 orT1R2, and modulation of taste transduction via GPCR activity is assayedby measuring changes in Ca2+ levels.

Methods of assaying for modulators of taste transduction include invitro ligand binding assays using receptors comprising T1R3, portionsthereof such as the extracellular domain, or chimeric proteinscomprising one or more domains of T1R3, and in in vivo (cell-based andanimal) assays such as oocyte T1R3 receptor expression; tissue culturecell T1R3 receptor expression; transcriptional activation of T1R3;phosphorylation and dephosphorylation of GPCRs; G-protein binding toGPCRs; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP and inositol triphosphate; changes inintracellular calcium levels; and neurotransmitter release.

DEFINITIONS

A “T1R family taste receptor” refers to a receptor comprising a memberof the T1R family of G-protein coupled receptors, e.g., T1R1, T1R2, andT1R3, or any combination thereof as a homodimer receptor, a heterodimerreceptor, or a monomer receptor. In one embodiment, the T1R familyreceptor comprises T1R3 (a “T1R3-comprising taste receptor” or a“T1R3-comprising sweet taste receptor”). In one embodiment, the T1Rfamily receptor comprises a first T1R3 polypeptide and a second T1R3polypeptide, which form a homodimeric receptor, either covalently ornon-covalently linked. In another embodiment, the T1R family receptorcomprises a single T1R3 polypeptide and no other T1R polypeptide, andforms a monomeric receptor. In another embodiment, the T1R familyreceptor comprises T1R3 and a heterologous polypeptide of the T1Rfamily. In one embodiment, the receptor comprises T1R1 and T1R3. Inanother embodiment, the receptor comprises T1R2 and T1R3. In oneembodiment the T1R3-comprising receptor is active when the two membersof the receptor are co-expressed in the same cell, e.g., T1R3 and T1R3,or T1R1 and T1R3 or T1R2 and T1R3. In another embodiment, the T1Rpolypeptides are co-expressed in the same cell and form a heterodimericor homodimeric receptor, in which the T1R polypeptides of the receptorare non-covalently linked or covalently linked. The receptor has theability to recognize, e.g., naturally occurring and/or artificial sweettasting molecule such as sucrose, fructose, galactose, mannose, glucose,lactose, saccharin, dulcin, acesulfame-K, as well as other molecules,sweet and non-sweet. These molecules are examples of compounds that“modulate sweet taste signal transduction” by acting as ligands for thetaste-transducing G protein coupled receptor comprising T1R3.

The terms “GPCR-B3 or T1R1,” “GPCR-B4 or T1R2,” and “T1R3” or a nucleicacid encoding “GPCR-B3 or T1R1,” “GPCR-B4 or T1R2,” and “T1R3” refer tonucleic acid and polypeptide polymorphic variants, alleles, mutants, andinterspecies homologs that are members of the T1R family of G proteincoupled receptors and: (1) have an amino acid sequence that has greaterthan about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%,90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity, preferably over a region of over a regionof at least about 25, 50, 100, 200, 500, 1000, or more amino acids, toan amino acid sequence encoded by SEQ ID NO:1, 2, 3, 7, 8, 9, 15, 18,20, 23, 25, 27, or 31; (2) bind to antibodies, e.g., polyclonalantibodies, raised against an immunogen comprising an amino acidsequence encoded by SEQ ID NO:1, 2, 3, 7, 8, 9, 15, 18, 20, 23, 25, 27,or 31, and conservatively modified variants thereof; (3) specificallyhybridize under stringent hybridization conditions to an anti-sensestrand corresponding to a nucleic acid sequence encoding a T1R protein,e.g., SEQ ID NO:4, 5, 6, 10, 11, 12, 13, 14, 16, 17, 19, 21, 22, 24, 26,28, or 30, and conservatively modified variants thereof; (4) have anucleic acid sequence that has greater than about 60% sequence identity,65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%, or higher nucleotide sequence identity, preferably overa region of at least about 25, 50, 100, 200, 500, 1000, or morenucleotides, to SEQ ID NO:4, 5, 6, 10, 11, 12, 13, 14, 16, 17, 19, 21,22, 24, 26, or 28, or 30. The T1R family polypeptide of the invention(e.g., T1R1, T1R2, or T1R3) or T1R3-comprising receptor (e.g., T1R3,T1R3+T1R3, T1R1+3 or T1R2+3) further has G protein coupled receptoractivity, either alone or when co-expressed in the same cell, or whenco-expressed as a monomer, homodimer, or heterodimer with another T1Rfamily member. Accession numbers for amino acid sequences and nucleotidesequences of human, rat, and mouse T1R1, T1R2, and T1R3 can be found inGenBank (for human T1R1 amino acid sequences, see, e.g., Accession No.DAA00012 and NP_(—)619642; for human T1R1 nucleotide sequences, see,e.g., Accession No. BK00153; for human T1R2 amino acid sequences, see,e.g., Accession No. DAA00019, AAM12239, and NP_(—)619642.1, for humanT1R2 nucleotide sequences, see, e.g., Accession No. BK000151,NM_(—)138697.1, AF458149S1-6; for human T1R3 amino acid sequences, see,e.g., Accession No. DAA00013, for human T1R3 nucleotide sequences, see,e.g., Accession NO. BK000152). See also WO 00/06592, WO 00/06593, WO01/66563, WO 03/001876, WO 02/064631, WO 03/004992, WO 03/025137, WO02/086079 and WO 01/83749 for amino acid and nucleotide sequences ofT1R1, T1R2, and T1R3, each herein incorporated by reference in itsentirety.

T1R proteins have “G-protein coupled receptor activity,” e.g., they bindto G-proteins in response to extracellular stimuli, such as ligandbinding (e.g., sweet ligands), and promote production of secondmessengers such as IP3, cAMP, and Ca2+ via stimulation of enzymes suchas phospholipase C and adenylate cyclase. Such activity can be measuredin a heterologous cell, by coupling a GPCR (or a chimeric GPCR) toeither a G-protein or promiscuous G-protein such as Gα15 or Gα₁₆-Gα_(z)and an enzyme such as PLC, and measuring increases in intracellularcalcium using (Offermans & Simon, J. Biol. Chem. 270:15175-15180(1995)). Receptor activity can be effectively measured, e.g., byrecording ligand-induced changes in [Ca²⁺]i using fluorescentCa²⁺-indicator dyes and fluorometric imaging.

Such GPCRs have transmembrane, extracellular and cytoplasmic domainsthat can be structurally identified using methods known to those ofskill in the art, such as sequence analysis programs that identifyhydrophobic and hydrophilic domains (see, e.g., Kyte & Doolittle, J.Mol. Biol. 157:105-132 (1982)). Such domains are useful for makingchimeric proteins and for in vitro assays of the invention (see, e.g.,WO 94/05695 and U.S. Pat. No. 5,508,384).

The phrase “functional effects” in the context of assays for testingcompounds that modulate activity (e.g., signal transduction) of a sweettaste receptor or protein of the invention includes the determination ofa parameter that is indirectly or directly under the influence of a GPCRor sweet taste receptor, e.g., a physical, phenotypic, or chemicaleffect, such as the ability to transduce a cellular signal in responseto external stimuli such as ligand binding, or the ability to bind aligand. It includes binding activity and signal transduction.“Functional effects” include in vitro, in vivo, and ex vivo activities.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a T1R GPCR protein or a sweet taste receptorcomprising one or more T1R GPCR proteins, e.g., physical and chemical orphenotypic effect. Such functional effects can be measured by any meansknown to those skilled in the art, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index);hydrodynamic (e.g., shape); chromatographic; or solubility propertiesfor the protein; measuring inducible markers or transcriptionalactivation of the protein; measuring binding activity or binding assays,e.g., binding to antibodies; measuring changes in ligand bindingactivity or analogs thereof, either naturally occurring or synthetic;measuring cellular proliferation; measuring cell surface markerexpression, measurement of changes in protein levels for T1R-associatedsequences; measurement of RNA stability; G-protein binding; GPCRphosphorylation or dephosphorylation; signal transduction, e.g.,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP3, PI, or intracellular Ca²⁺); neurotransmitter release;hormone release; voltage, membrane potential and conductance changes;ion flux; regulatory molecule binding; identification of downstream orreporter gene expression (CAT, luciferase, β-gal, GFP and the like),e.g., via chemiluminescence, fluorescence, colorimetric reactions,antibody binding, and inducible markers.

“Inhibitors,” “activators,” and “modulators” of T1R familypolynucleotide and polypeptide sequences and T1R family taste receptorsare used to refer to activating, inhibitory, or modulating moleculesidentified using in vitro and in vivo assays of T1R polynucleotide andpolypeptide sequences and T1R family taste receptors, includingmonomeric, homodimeric and heterodimeric receptors. Inhibitors arecompounds that, e.g., bind to, partially or totally block activity,decrease, prevent, delay activation, inactivate, desensitize, or downregulate the activity or expression of the T1R family of taste receptorssuch as a receptor comprising a T1R3 polypeptide, e.g., antagonists.“Activators” are compounds that increase, open, activate, facilitate,enhance activation, sensitize, agonize, or up regulate a T1R familytaste receptor, such as a receptor comprising a T1R3 polypeptide, e.g.,agonists. Inhibitors, activators, or modulators also include geneticallymodified versions of T1R family taste receptors, e.g., versions withaltered activity, as well as naturally occurring and synthetic ligands,antagonists, agonists, antibodies, antisense molecules, ribozymes, smallchemical molecules and the like. Such assays for inhibitors andactivators include, e.g., expressing T1R family taste receptors invitro, in cells, or cell membranes, applying putative modulatorcompounds, and then determining the functional effects on activity, asdescribed above. In one embodiment, taste receptor comprising a T1R3polypeptide has the ability to recognize a sweet tasting molecule suchas sucrose, glucose, fructose, lactose, mannose, galactose, saccharin,dulcin, acesulfame-K. In another embodiment, a taste receptor comprisinga T1R3 polypeptide has the ability to recognize other molecules, such aspotential artificial sweeteners. These molecules are examples ofcompounds that modulate taste signal transduction by acting asextracellular ligands for the G protein coupled receptor and activatingthe receptor. In other embodiments, compounds that modulate taste signaltransduction are molecules that act as intracellular ligands of thereceptor, or inhibit or activate binding of an extracellular ligand, orinhibit or activate binding of intracellular ligands of the receptor.

Samples or assays comprising the T1R family of taste receptors aretreated with a potential activator, inhibitor, or modulator are comparedto control samples without the inhibitor, activator, or modulator toexamine the extent of inhibition. Control samples (untreated withinhibitors) are assigned a relative protein activity value of 100%.Inhibition of a T1R family receptor is achieved when the activity valuerelative to the control is about 80%, preferably 50%, more preferably25-0%. Activation of a T1R family receptor is achieved when the activityvalue relative to the control (untreated with activators) is 110%, morepreferably 150%, more preferably 200-500% (i.e., two to five fold higherrelative to the control), more preferably 1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, such asan artificial sweetener or naturally occurring sugar, either naturallyoccurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5to about 25 amino acids in length, preferably from about 10 to 20 or 12to 18 amino acids in length, preferably 12, 15, or 18 amino acids inlength), small organic molecule, polysaccharide, lipid, fatty acid,polynucleotide, oligonucleotide, etc., to be tested for the capacity todirectly or indirectly modulation taste. The test compound can be in theform of a library of test compounds, such as a combinatorial orrandomized library that provides a sufficient range of diversity. Testcompounds are optionally linked to a fusion partner, e.g., targetingcompounds, rescue compounds, dimerization compounds, stabilizingcompounds, addressable compounds, and other functional moieties.Conventionally, new chemical entities with useful properties aregenerated by identifying a test compound (called a “lead compound”) withsome desirable property or activity, e.g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Biological sample” include sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, sputum, tissue, cultured cells, e.g., primarycultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

A “heterodimer” is a dimer receptor comprising two different polypeptidesubunits, e.g., two different polypeptides, where the molecules areassociated via either covalent, e.g., through a linker or a chemicalbond, or non-covalent, e.g., ionic, van der Waals, electrostatic, orhydrogen bonds linkages. The T1R3-comprising receptors of the inventionfunction when co-expressed in the same cell, preferably whenco-expressed so that they form a heterodimer, either covalently ornon-covalently linked. For example, T1R1 and T1R3 form a heteromericreceptor, and T1R2 and T1R3 form a heteromeric receptor.

A “homodimer” is a dimer receptor comprising two of the same polypeptidesubunits, e.g., two T1R3 polypeptides, where the molecules areassociated via either covalent, e.g., through a linker or a chemicalbond, or non-covalent, e.g., ionic, van der Waals, electrostatic, orhydrogen bonds linkages. The T1R3-comprising receptors of the inventionfunction when co-expressed in the same cell, preferably whenco-expressed so that they form a homodimer, either covalently ornon-covalently linked.

A “monomer” is a receptor comprising one polypeptide subunit, e.g., oneT1R3 polypeptide.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., nucleotide sequences SEQ ID NO:1-25), whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, enantiomers (D- and L-forms), and achiral amino acids, as well asamino acid analogs and amino acid mimetics that function in a mannersimilar to the naturally occurring amino acids. Naturally occurringamino acids are those encoded by the genetic code, as well as thoseamino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that functions in a manner similar to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains, e.g., extracellulardomains, transmembrane domains, and cytoplasmic domains. Domains areportions of a polypeptide that form a compact unit of the polypeptideand are typically 15 to 350 amino acids long. Typical domains are madeup of sections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, as well asthe complements of any such sequence. Also included are DNA, cDNA, RNA,polynucleotides, nucleotides, and the like. The term encompasses nucleicacids containing known nucleotide analogs or modified backbone residuesor linkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5× SSC, and 1% SDS, incubating at 42° C., or,5× SSC, 1% SDS, incubating at 65° C., with wash in 0.2× SSC, and 0.1%SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1× SSC at 45° C. A positive hybridization is at leasttwice background. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include 30-40cycles of the following conditions: a denaturation phase of 90° C.-95°C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and anextension phase of about 72° C. for 1-2 min. Protocols and guidelinesfor low and high stringency amplification reactions are provided, e.g.,in Innis et al., PCR Protocols, A Guide to Methods and Applications(1990).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). Techniques for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to a T1Rprotein or a homodimeric or heterodimeric T1R3-comprising taste receptorcomprising a sequence of or encoded by SEQ ID NO:1-25, polymorphicvariants, alleles, orthologs, and conservatively modified variants, orsplice variants, or portions thereof, can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withT1R proteins and/or homodimeric or heterodimeric T1R3-comprising tastereceptors and not with other proteins. In one embodiment, the antibodiesreact with a homodimeric T1R3-comprising taste receptor, but not withindividual protein members of the T1R family. This selection may beachieved by subtracting out antibodies that cross-react with othermolecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

Isolation of Nucleic Acids Encoding T1R Family Members

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

T1R nucleic acids, polymorphic variants, orthologs, and alleles that aresubstantially identical to an amino acid sequences disclosed herein canbe isolated using T1R nucleic acid probes and oligonucleotides understringent hybridization conditions, by screening libraries.Alternatively, expression libraries can be used to clone T1R protein,polymorphic variants, orthologs, and alleles by detecting expressedhomologs immunologically with antisera or purified antibodies madeagainst human T1R or portions thereof.

To make a cDNA library, one should choose a source that is rich in T1RRNA, e.g., taste buds such as circumvallate, foliate, fungiform, andpalate. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25:263-269 (1983); Sambrook et al, supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating T1R nucleic acid and its orthologs,alleles, mutants, polymorphic variants, and conservatively modifiedvariants combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of human T1R directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify T1R homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of T1R encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of T1R can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology, e.g., and the like.

Nucleic acids encoding T1R protein can be used with high densityoligonucleotide array technology (e.g., GeneChip™) to identify T1Rprotein, orthologs, alleles, conservatively modified variants, andpolymorphic variants in this invention (see, e.g., Gunthand et al., AIDSRes. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med.2:753-759 (1996); Matson et al., Anal Biochem. 224:110-106 (1995);Lockhart et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al.,Genome Res. 8:435-448 (1998); Hacia et al., Nucleic Acids Res.26:3865-3866 (1998)).

The gene for T1R is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding a T1R protein, one typically subclones T1R into an expressionvector that contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation. The T1Rnucleic acids can be co-expressed or separately expressed, preferablyco-expressed on the same or a different vector. Suitable bacterialpromoters are well known in the art and described, e.g., in Sambrook etal., and Ausubel et al, supra. Bacterial expression systems forexpressing the T1R protein are available in, e.g., E. coli, Bacillussp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach etal., Nature 302:543-545 (1983). Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available. In one preferred embodiment, retroviralexpression systems are used in the present invention.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the T1R encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding T1R andsignals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, β-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal.

In one embodiment, the vectors of the invention have a regulatablepromoter, e.g., tet-regulated systems and the RU-486 system (see, e.g.,Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547 (1992); Oligino etal., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441(1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al.,Nat. Biotechnol. 16:757-761 (1998)). These impart small molecule controlon the expression of the candidate target nucleic acids. This beneficialfeature can be used to determine that a desired phenotype is caused by atransfected cDNA rather than a somatic mutation.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with a T1Rencoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of T1R protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingT1R.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofT1R, which is recovered from the culture using standard techniquesidentified below.

Purification of T1R Polypeptides

Either naturally occurring or recombinant T1R polypeptides orT1R3-comprising receptors can be purified for use in functional assays.Naturally occurring T1R proteins or T1R3-comprising receptors can bepurified, e.g., from human tissue. Recombinant T1R proteins orT1R3-comprising receptors can be purified from any suitable expressionsystem. T1R polypeptides are typically co-expressed in the same cell toform T1R3-comprising receptors.

The T1R protein or T1R3-comprising receptor may be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).

A number of procedures can be employed when recombinant T1R protein orT1R3-comprising receptor is being purified. For example, proteins havingestablished molecular adhesion properties can be reversible fused to theT1R protein or T1R3-comprising receptor. With the appropriate ligand,T1R protein or T1R3-comprising receptor can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,T1R protein or T1R3-comprising receptor could be purified usingimmunoaffinity columns.

A. Purification of T1R from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of T1R proteinor T1R3-comprising receptor inclusion bodies. For example, purificationof inclusion bodies typically involves the extraction, separation and/orpurification of inclusion bodies by disruption of bacterial cells, e.g.,by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mMMgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can belysed using 2-3 passages through a French Press, homogenized using aPolytron (Brinkman Instruments) or sonicated on ice. Alternate methodsof lysing bacteria are apparent to those of skill in the art (see, e.g.,Sambrook et al., supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human T1R proteins orT1R3-comprising receptors are separated from other bacterial proteins bystandard separation techniques, e.g., with Ni—NTA agarose resin.

Alternatively, it is possible to purify T1R protein or T1R3-comprisingreceptor from bacteria periplasm. After lysis of the bacteria, when theT1R protein or T1R3-comprising receptor is exported into the periplasmof the bacteria, the periplasmic fraction of the bacteria can beisolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

B. Standard Protein Separation Techniques for Purifying T1R ProteinsSolubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the T1R proteins or T1R3-comprising receptorscan be used to isolate it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column Chromatography

The T1R proteins or T1R3-comprising receptors can also be separated fromother proteins on the basis of its size, net surface charge,hydrophobicity, and affinity for ligands. In addition, antibodies raisedagainst proteins can be conjugated to column matrices and the proteinsimmunopurified. All of these methods are well known in the art. It willbe apparent to one of skill that chromatographic techniques can beperformed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

Assays for Modulators of T1R Protein

A. Assays

Modulation of a T1R3-comprising taste receptor, and correspondingmodulation of taste, can be assessed using a variety of in vitro and invivo assays. Such assays can be used to test for inhibitors andactivators of T1R3-comprising taste receptors, and, consequently,inhibitors and activators of taste. Such modulators of T1R3-comprisingsweet taste receptors, which are involved in taste signal transduction.Modulators of T1R3-comprising taste receptors are tested using eitherrecombinant or naturally occurring T1R3-comprising taste receptors,preferably human receptors.

In one embodiment, the monomeric or homodimeric T1R3-comprisingreceptors of the invention can be used to screen for naturally occurringor artificial sweet tasting molecules, e.g., small organic molecules,amino acids, peptides, carbohydrates, lipids, polysaccharides, etc. Forexample, homodimeric or monomeric T1R3-comprising receptors of theinvention recognize naturally occurring sweet tastants, as describedbelow in the example section. Such receptors can be used to screen forartificial sweeteners, or altered naturally occurring sweeteners, thatmimic the naturally occurring sugar ligands of the homodimeric ormonomeric T1R3-comprising receptor.

Preferably, the T1R3-comprising taste receptor will have a sequence asencoded by a sequence provided herein or a conservatively modifiedvariant thereof. Alternatively, the T1R3-comprising taste receptor ofthe assay will be derived from a eukaryote and include an amino acidsubsequence having substantial amino acid sequence identity to thesequences provided herein or is encoded by a nucleotide sequence thathybridizes under stringent conditions (moderate or high) to a nucleotidesequence as described herein. Generally, the amino acid sequenceidentity will be at least 60%, preferably at least 65%, 70%, 75%, 80%,85%, or 90%, most preferably at least 95%.

Measurement of sweet taste signal transduction or loss-of-sweet tastesignal transduction phenotype on T1R3-comprising taste receptor or cellexpressing the T1R3-comprising taste receptor, either recombinant ornaturally occurring, can be performed using a variety of assays, invitro, in vivo, and ex vivo, as described herein. A suitable physical,chemical or phenotypic change that affects activity or binding can beused to assess the influence of a test compound on the polypeptide ofthis invention. When the functional effects are determined using intactcells or animals, one can also measure a variety of effects such as, inthe case of signal transduction, e.g., ligand binding, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., northern blots), changes in cell metabolism such as pHchanges, and changes in intracellular second messengers such as Ca²⁺,IP3, cGMP, or cAMP.

In Vitro Assays

Assays to identify compounds with T1R3-comprising taste receptormodulating activity can be performed in vitro. Such assays can use afull length T1R3-comprising taste receptor or a variant thereof, or afragment of a T1R3-comprising taste receptor, such as an extracellulardomain, fused to a heterologous protein to form a chimera (see, e.g., WO01/66563, WO 03/001876, WO 02/064631, and WO 03/004992). Purifiedrecombinant or naturally occurring T1R3-comprising taste receptor can beused in the in vitro methods of the invention. In addition to purifiedT1R3-comprising taste receptor, the recombinant or naturally occurringT1R3-comprising taste receptor can be part of a cellular lysate or acell membrane. As described below, the binding assay can be either solidstate or soluble. Preferably, the protein or membrane is bound to asolid support, either covalently or non-covalently. Often, the in vitroassays of the invention are ligand binding or ligand affinity assays,either non-competitive or competitive (with known extracellular ligandsas described herein, or with a known intracellular ligand GTP). Other invitro assays include measuring changes in spectroscopic (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the protein.

In one embodiment, a high throughput binding assay is performed in whichthe T1R3-comprising taste receptor or chimera comprising a fragmentthereof is contacted with a potential modulator and incubated for asuitable amount of time. In one embodiment, the potential modulator isbound to a solid support, and the T1R3-comprising taste receptor isadded. In another embodiment, the T1R3-comprising taste receptor isbound to a solid support. A wide variety of modulators can be used, asdescribed below, including small organic molecules, peptides,antibodies, and T1R3-comprising taste receptor ligand analogs. A widevariety of assays can be used to identify T1R3-comprising tastereceptor-modulator binding, including labeled protein-protein bindingassays, electrophoretic mobility shifts, immunoassays, enzymatic assayssuch as phosphorylation assays, and the like. In some cases, the bindingof the candidate modulator is determined through the use of competitivebinding assays, where interference with binding of a known ligand ismeasured in the presence of a potential modulator. Ligands forT1R3-comprising taste receptors are provided herein. Either themodulator or the known ligand is bound first, and then the competitor isadded. After the T1R3-comprising taste receptor is washed, interferencewith binding, either of the potential modulator or of the known ligand,is determined. Often, either the potential modulator or the known ligandis labeled.

Cell-based In Vivo Assays

In another embodiment, a T1R3-comprising taste receptor is expressed ina cell (e.g., by expression or co-expression one or two members of theT1R family such as T1R1 and T1R3 or T1R2 and T1R3, preferably byexpression of T1R3 alone without expression of any other T1R familymembers), and functional, e.g., physical and chemical or phenotypic,changes are assayed to identify T1R3-comprising taste receptor tastemodulators. Cells expressing T1R3-comprising taste receptor can also beused in binding assays. Any suitable functional effect can be measured,as described herein. For example, ligand binding, G-protein binding, andGPCR signal transduction, e.g., changes in intracellular Ca²⁺ levels,are all suitable assays to identify potential modulators using a cellbased system. Suitable cells for such cell based assays include bothprimary cells and cell lines, as described herein. The T1R3-comprisingtaste receptor can be naturally occurring or recombinant. Also, asdescribed above, chimeric T1R3-comprising taste receptors with GPCRactivity can be used in cell based assays. For example, theextracellular domain of an T1R protein can be fused to the transmembraneand/or cytoplasmic domain of a heterologous protein, preferably aheterologous GPCR. Such a chimeric GPCR would have GPCR activity andcould be used in cell based assays of the invention.

In another embodiment, cellular T1R polypeptide levels are determined bymeasuring the level of protein or mRNA. The level of T1R protein orproteins related to T1R signal transduction are measured usingimmunoassays such as western blotting, ELISA and the like with anantibody that selectively binds to the T1R3-comprising taste receptor ora fragment thereof. For measurement of mRNA, amplification, e.g., usingPCR, LCR, or hybridization assays, e.g., northern hybridization, RNAseprotection, dot blotting, are preferred. The level of protein or mRNA isdetected using directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

Alternatively, T1R3-comprising receptor expression can be measured usinga reporter gene system. Such a system can be devised using an T1Rprotein promoter operably linked to a reporter gene such aschloramphenicol acetyltransferase, firefly luciferase, bacterialluciferase, β-galactosidase and alkaline phosphatase. Furthermore, theprotein of interest can be used as an indirect reporter via attachmentto a second reporter such as red or green fluorescent protein (see,e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)). Thereporter construct is typically transfected into a cell. After treatmentwith a potential modulator, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

In another embodiment, a functional effect related to GPCR signaltransduction can be measured. An activated or inhibited T1R3-comprisingG-coupled protein receptor will alter the properties of target enzymes,second messengers, channels, and other effector proteins. The examplesinclude the activation of cGMP phosphodiesterase, adenylate cyclase,phospholipase C, IP3, and modulation of diverse channels by G proteins.Downstream consequences can also be examined such as generation ofdiacyl glycerol and IP3 by phospholipase C, and in turn, for calciummobilization by IP3. Activated GPCR receptors become substrates forkinases that phosphorylate the C-terminal tail of the receptor (andpossibly other sites as well). Thus, activators will promote thetransfer of ³²P from gamma-labeled GTP to the receptor, which can beassayed with a scintillation counter. The phosphorylation of theC-terminal tail will promote the binding of arrestin-like proteins andwill interfere with the binding of G-proteins. For a general review ofGPCR signal transduction and methods of assaying signal transduction,see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96(1983); Bourne et al., Nature 10:349:117-27 (1991); Bourne et al.,Nature 348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem. 67:653-92(1998).

As described above, activation of some G-protein coupled receptorsstimulates the formation of inositol triphosphate (IP3) throughphospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge &Irvine, Nature 312:315-21 (1984)). IP3 in turn stimulates the release ofintracellular calcium ion stores. Thus, a change in cytoplasmic calciumion levels, or a change in second messenger levels such as IP3 can beused to assess G-protein coupled receptor function. Cells expressingsuch G-protein coupled receptors may exhibit increased cytoplasmiccalcium levels as a result of contribution from both intracellularstores and via activation of ion channels, in which case it may bedesirable although not necessary to conduct such assays in calcium-freebuffer, optionally supplemented with a chelating agent such as EGTA, todistinguish fluorescence response resulting from calcium release frominternal stores.

In one example, T1R3-comprising taste receptor GPCR activity is measuredby expressing a T1R3-comprising taste receptor in a heterologous cellwith a promiscuous G-protein that links the receptor to a phospholipaseC signal transduction pathway (see Offermanns & Simon, J. Biol. Chem.270:15175-15180 (1995)). Modulation of signal transduction is assayed bymeasuring changes in intracellular Ca²⁺ levels, which change in responseto modulation of the GPCR signal transduction pathway via administrationof a molecule that associates with an T1R3-comprising taste receptor.Changes in Ca²⁺ levels are optionally measured using fluorescent Ca²⁺indicator dyes and fluorometric imaging.

In another example, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which may or may not contain an agonist).

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. In cases where activation of the receptorresults in a decrease in cyclic nucleotide levels, it may be preferableto expose the cells to agents that increase intracellular cyclicnucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay.

In one example, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon,J. Biol. Chem. 270:15175-15180 (1995) may be used to determine the levelof cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp.Cell and Mol. Biol. 11:159-164 (1994) may be used to determine the levelof cGMP. Further, an assay kit for measuring cAMP and/or cGMP isdescribed in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In one example, assays for G-protein coupled receptor activity includecells that are loaded with ion or voltage sensitive dyes to reportreceptor activity. Assays for determining activity of such receptors canalso use known agonists and antagonists for other G-protein coupledreceptors as negative or positive controls to assess activity of testedcompounds. In assays for identifying modulatory compounds (e.g.,agonists, antagonists), changes in the level of ions in the cytoplasm ormembrane voltage will be monitored using an ion sensitive or membranevoltage fluorescent indicator, respectively. Among the ion-sensitiveindicators and voltage probes that may be employed are those disclosedin the Molecular Probes 1997 Catalog. For G-protein coupled receptors,promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay ofchoice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053(1991)). Such promiscuous G-proteins allow coupling of a wide range ofreceptors.

Animal Models

Animal models of taste also find use in screening for modulators oftaste, such as the T1R knockout mouse strains as described herein.Transgenic animal technology including gene knockout technology, forexample as a result of homologous recombination with an appropriate genetargeting vector, or gene overexpression, will result in the absence orincreased expression of the T1R3-comprising receptor or componentsthereof. When desired, tissue-specific expression or knockout of theT1R3-comprising receptors or components thereof may be necessary.Transgenic animals generated by such methods find use as animal modelsof taste modulation and are additionally useful in screening formodulators of taste modulation.

B. Modulators

The compounds tested as modulators of T1R3-comprising taste receptorscan be any small organic molecule, or a biological entity, such as aprotein, e.g., an antibody or peptide, an amino acid, a lipid, a fat, asugar, e.g., a mono-, di-, or polysaccharide, a nucleic acid, e.g., anantisense oligonucleotide or a ribozyme, or a small organic molecule.Alternatively, modulators can be genetically altered versions of aT1R3-comprising taste receptor. Typically, test compounds will be smallorganic molecules, amino acids, peptides, lipids, and mono-, di- andpolysaccharides.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial small organic molecule or peptide librarycontaining a large number of potential therapeutic compounds (potentialmodulator or ligand compounds). Such “combinatorial chemical libraries”or “ligand libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using aT1R3-comprising taste receptor, or a cell or tissue expressing aT1R3-comprising taste receptor, either naturally occurring orrecombinant. In another embodiment, the invention provides solid phasebased in vitro assays in a high throughput format, where theT1R3-comprising taste receptor is attached to a solid phase substrate.Any one of the assays described herein can be adapted for highthroughput screening, e.g., ligand binding, cellular proliferation, cellsurface marker flux, e.g., screening, radiolabeled GTP binding, secondmessenger flux, e.g., Ca²⁺, IP3, cGMP, or cAMP, cytokine production,etc.

In the high throughput assays of the invention, either soluble or solidstate, it is possible to screen up to several thousand differentmodulators or ligands in a single day. This methodology can be used forT1R3-comprising taste receptors in vitro, or for cell-based ormembrane-based assays comprising T1R3-comprising taste receptors. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100−about 1500different compounds. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds are possible using the integrated systems of theinvention.

For a solid state reaction, the protein of interest or a fragmentthereof, e.g., an extracellular domain, or a cell or membrane comprisingthe protein of interest or a fragment thereof as part of a fusionprotein can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly Gly sequencesof between about 5 and 200 amino acids (SEQ ID NO:32). Such flexiblelinkers are known to persons of skill in the art. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc. Huntsville, Ala. These Linkers optionally have amide linkage,sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Immunological Detection of T1R3-Comprising Receptors

In addition to the detection of T1R genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect T1R3-comprising taste receptors of the invention. Such assays areuseful for screening for modulators of T1R3-comprising taste receptors,as well as for therapeutic and diagnostic applications. Immunoassays canbe used to qualitatively or quantitatively analyze T1R3-comprising tastereceptors. A general overview of the applicable technology can be foundin Harlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Production of Antibodies

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the T1R proteins and T1R3-comprising taste receptorsare known to those of skill in the art (see, e.g., Coligan, CurrentProtocols in Immunology (1991); Harlow & Lane, supra; Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986); and Kohler &Milstein, Nature 256:495-497 (1975). Such techniques include antibodypreparation by selection of antibodies from libraries of recombinantantibodies in phage or similar vectors, as well as preparation ofpolyclonal and monoclonal antibodies by immunizing rabbits or mice (see,e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature341:544-546 (1989)).

A number of immunogens comprising portions of T1R protein orT1R3-comprising taste receptor may be used to produce antibodiesspecifically reactive with T1R protein. For example, recombinant T1Rprotein or an antigenic fragment thereof, can be isolated as describedherein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-T1R orT1R3-comprising taste receptor proteins, using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better. Antibodies specific only for a particularT1R3-comprising taste receptor ortholog, such as human T1R3-comprisingtaste receptor, can also be made, by subtracting out othercross-reacting orthologs from a species such as a non-human mammal. Inaddition, individual T1R proteins can be used to subtract out antibodiesthat bind both to the receptor and the individual T1R proteins. In thismanner, antibodies that bind only to a particular receptor may beobtained.

Once the specific antibodies against T1R3-comprising taste receptors areavailable, the protein can be detected by a variety of immunoassaymethods. In addition, the antibody can be used therapeutically as aT1R3-comprising taste receptor modulators. For a review of immunologicaland immunoassay procedures, see Basic and Clinical Immunology (Stites &Terr eds., 7^(th) ed. 1991). Moreover, the immunoassays of the presentinvention can be performed in any of several configurations, which arereviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); andHarlow & Lane, supra.

B. Immunological Binding Assays

T1R3-comprising taste receptors can be detected and/or quantified usingany of a number of well recognized immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Methods in CellBiology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basicand Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casethe T1R3-comprising taste receptor or antigenic subsequence thereof).The antibody (e.g., anti-T1R3-comprising taste receptor) may be producedby any of a number of means well known to those of skill in the art andas described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled T1R3-comprising tastereceptor or a labeled anti-T1R3-comprising taste receptor antibody.Alternatively, the labeling agent may be a third moiety, such asecondary antibody, that specifically binds to theantibody/T1R3-comprising taste receptor complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). Thelabeling agent can be modified with a detectable moiety, such as biotin,to which another molecule can specifically bind, such as streptavidin. Avariety of detectable moieties are well known to those skilled in theart.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting T1R3-comprising taste receptors in samplesmay be either competitive or noncompetitive. Noncompetitive immunoassaysare assays in which the amount of antigen is directly measured. In onepreferred “sandwich” assay, for example, the anti-T1R3-comprising tastereceptor antibodies can be bound directly to a solid substrate on whichthey are immobilized. These immobilized antibodies then captureT1R3-comprising taste receptors present in the test sample.T1R3-comprising taste receptors thus immobilized are then bound by alabeling agent, such as a second T1R3-comprising taste receptor antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond or third antibody is typically modified with a detectable moiety,such as biotin, to which another molecule specifically binds, e.g.,streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of T1R3-comprising taste receptorpresent in the sample is measured indirectly by measuring the amount ofa known, added (exogenous) T1R3-comprising taste receptor displaced(competed away) from an anti-T1R3-comprising taste receptor antibody bythe unknown T1R3-comprising taste receptor present in a sample. In onecompetitive assay, a known amount of T1R3-comprising taste receptor isadded to a sample and the sample is then contacted with an antibody thatspecifically binds to a T1R3-comprising taste receptor. The amount ofexogenous T1R3-comprising taste receptor bound to the antibody isinversely proportional to the concentration of T1R3-comprising tastereceptor present in the sample. In a particularly preferred embodiment,the antibody is immobilized on a solid substrate. The amount ofT1R3-comprising taste receptor bound to the antibody may be determinedeither by measuring the amount of T1R3-comprising taste receptor presentin a T1R3-comprising taste receptor/antibody complex, or alternativelyby measuring the amount of remaining uncomplexed protein. The amount ofT1R3-comprising taste receptor may be detected by providing a labeledT1R3-comprising taste receptor molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known T1R3-comprising taste receptor is immobilized on asolid substrate. A known amount of anti-T1R3-comprising taste receptorantibody is added to the sample, and the sample is then contacted withthe immobilized T1R3-comprising taste receptor. The amount ofanti-T1R3-comprising taste receptor antibody bound to the knownimmobilized T1R3-comprising taste receptor is inversely proportional tothe amount of T1R3-comprising taste receptor present in the sample.Again, the amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a T1R3-comprising tastereceptor can be immobilized to a solid support. Proteins (e.g.,T1R3-comprising taste receptors and homologs) are added to the assaythat compete for binding of the antisera to the immobilized antigen. Theability of the added proteins to compete for binding of the antisera tothe immobilized protein is compared to the ability of theT1R3-comprising taste receptor to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of aT1R3-comprising taste receptor, to the immunogen protein. In order tomake this comparison, the two proteins are each assayed at a wide rangeof concentrations and the amount of each protein required to inhibit 50%of the binding of the antisera to the immobilized protein is determined.If the amount of the second protein required to inhibit 50% of bindingis less than 10 times the amount of the T1R3-comprising taste receptorthat is required to inhibit 50% of binding, then the second protein issaid to specifically bind to the polyclonal antibodies generated to aT1R3-comprising taste receptor immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of T1R3-comprising taste receptors in the sample. The techniquegenerally comprises separating sample proteins by gel electrophoresis onthe basis of molecular weight, transferring the separated proteins to asuitable solid support, (such as a nitrocellulose filter, a nylonfilter, or derivatized nylon filter), and incubating the sample with theantibodies that specifically bind T1R3-comprising taste receptors. Theanti-T1R3-comprising taste receptor antibodies specifically bind to theT1R3-comprising taste receptor on the solid support. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the anti-T1R3-comprising taste receptorantibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize T1R3-comprisingtaste receptors, or secondary antibodies that recognizeanti-T1R3-comprising taste receptor.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Pharmaceutical Compositions and Administration

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid,oligonucleotide, amino acid, protein, peptide, small organic molecule,lipid, carbohydrate, mono-, di- or polysaccharide, particle, ortransduced cell), as well as by the particular method used to administerthe composition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed., 1989).Administration can be in any convenient manner, e.g., by injection, oraladministration, inhalation, transdermal application, or rectaladministration.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of a T1R3-comprising taste receptor, the physicianevaluates circulating plasma levels of the vector, vector toxicities,progression of the disease, and the production of anti-vectorantibodies. In general, the dose equivalent of a naked nucleic acid froma vector is from about 1 μg to 100 μg for a typical 70 kilogram patient,and doses of vectors which include a retroviral particle are calculatedto yield an equivalent amount of therapeutic nucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of T1R3-comprisingtaste receptors for the transfection of cells in vitro and in vivo.These nucleic acids can be inserted into any of a number of well-knownvectors for the transfection of target cells and organisms as describedbelow. The nucleic acids are transfected into cells, ex vivo or in vivo,through the interaction of the vector and the target cell. The nucleicacid, under the control of a promoter, then expresses a T1R3-comprisingtaste receptor of the present invention, by co-expressing two members ofthe T1R family, thereby mitigating the effects of absent, partialinactivation, or abnormal expression of a T1R3-comprising tastereceptor. The compositions are administered to a patient in an amountsufficient to elicit a therapeutic response in the patient. An amountadequate to accomplish this is defined as “therapeutically effectivedose or amount.”

Such gene therapy procedures have been used to correct acquired andinherited genetic defects and other diseases in a number of contexts.The ability to express artificial genes in humans facilitates theprevention and/or cure of many important human diseases, including manydiseases which are not amenable to treatment by other therapies (for areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1998); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology (Doerfler & Böhm eds., 1995); and Yu et al.,Gene Therapy 1:13-26 (1994)).

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EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

Results

Generation of T1R1, T1R2 and T1R3 KO mice

Expression of T1R receptors defines three largely non-overlappingpopulations of taste cells in the tongue and palate: cells co-expressingT1R1 and T1R3 (T1R1+3), cells co-expressing T1R2 and T1R3 (T1R2+3), andcells expressing T1R3 alone (Nelson, G. et al., Cell, 106, 381-390(2001)). Heterologous expression studies of T1Rs in HEK cellsdemonstrated that T1R1 and T1R3 combine to form a broadly tuned L-aminoacid receptor, while co-expression of T1R2 and T1R3 generates a sweettaste receptor that responds to all classes of sweet-tasting compounds(Nelson, G. et al., Cell, 106, 381-390 (2001); Nelson, G. et al.,Nature, 416, 199-202 (2002); Li, X. et al., Proc Natl Acad Sci USA, 99,4692-4696 (2002)). If T1R3 functions in vivo as a common component ofthe sweet and amino acid taste receptors, then a knockout of this GPCRshould generate mice devoid of sweet and amino acid taste reception. Incontrast, knockout of T1R1 or T1R2 might be expected to selectivelyaffect a single taste modality.

To define the role of T1Rs in vivo, we generated knockout mice that lackeach of the T1Rs by deleting exons encoding domains essential forreceptor function. FIG. 1 illustrates the KO strategies and shows insitu hybridization experiments demonstrating a complete lack of specificT1R staining in the corresponding homozygous KO animals. In order toensure that loss of any one T1R did not affect the viability orintegrity of taste cells, we also compared the expression of other T1Rs,T2Rs, PLCb2 (Rossler, P. et al., Eur J Cell Biol, 77, 253-261 (1998);Zhang, Y. et al., Cell, 112, 293-301 (2003)) and TRPM5 (Perez, C. A. etal., Nat Neurosci, 5, 1169-1176 (2002); Zhang, Y. et al., Cell, 112,293-301 (2003)) in control and KO animals. No significant differenceswere observed in the number or distribution of T1R-, T2R, PLCb2 andTRPM5-positive cells between wild type and KO taste tissue (FIG. 1 anddata not shown).

Two complementary strategies were used to assay the taste responses ofthe genetically modified mice. First, we recorded tastant-induced actionpotentials from one of the major nerves innervating taste receptor cellsof the tongue (chorda tympani). This physiological assay monitors theactivity of the taste system at the periphery, and provides a measure oftaste receptor cell function. Second, we examined taste behavior bymeasuring taste-choices in standard long-term two-bottle intakepreference assays, or by direct counting of immediate licking responsesin a multi-channel gustometer (Glendinning, J. I. et al., Chem Senses,27, 461-474 (2002); Zhang, Y. et al., Cell, 112, 293-301 (2003); seeExperimental Procedures). This second method relies on very shortexposures to tastants (5 s events over a total of 30 min versus 48 hrsfor two-bottle preference assays), and therefore has the great advantageof minimizing the impact of other sensory inputs, and post-ingestive andlearning effects from the assay.

FIG. 2 shows that knockouts of T1Rs have no significant effect either onphysiological or behavioral responses to citric acid, sodium chloride,and a variety of bitter tastants. These results demonstrate that bitter,salty and sour taste reception and perception operate through pathwaysindependent of T1R receptors, and further substantiate a model of codingat the periphery in which individual modalities operate independently ofeach other.

T1R1+3 is the Umami Receptor

Previously, Chaudhari et al described a truncated variant of themetabotropic glutamate receptor4 (mGluR4t) and suggested that itfunctions as the umami taste receptor (Chaudhari, N. et al., NatNeurosci, 3, 113-119 (2000)). We find this proposal unsatisfactory formany reasons. (1) The mGluR4t variant is missing the mGluR4 signalsequence needed for surface targeting. (2) This putative receptor alsolacks large fractions of the domains essential for glutamate recognitionas revealed by the crystal structure of the glutamate binding domain ofmGluR (Kunishima, N., et al., Nature, 407, 971-977 (2000)). (3) mGluR4tumami signaling has been proposed to operate via a cAMP pathway (Abaffy,T. et al., Am J Physiol Cell Physiol, 284, C1420-1428 (2003); Chaudhari,N. et al., Nat Neurosci, 3, 113-119 (2000)). However, amino acid/umamitaste is a PLCβ2/TRPM5-dependent process (Zhang, Y. et al., Cell, 112,293-301 (2003)). (4) Umami taste, but not mGluR4 activity, is stronglyaffected by the umami enhancers IMP and GMP. (5) Finally, mGluR4 KOanimals retain responses to umami stimuli (Chaudhari, N., and Roper, S.D., Ann N Y Acad Sci, 855, 398-406 (1998)). In contrast, recent evidencesuggest that the T1R1+3 amino acid receptor may function as themammalian umami (glutamate) taste sensor: First, the human and rodentT1R1+3 receptors display selectivity and sensitivity differences thatmimic amino acid taste differences between rodents and humans (Nelson,G. et al., Nature, 416, 199-202 (2002); Yoshii et al., 1986). Second,T1R1+3 activity is reliably enhanced by IMP and GMP, the two best knownpotentiators of umami taste in vivo (Nelson, G. et al., Nature, 416,199-202 (2002); Li, X. et al., Proc Natl Acad Sci USA, 99, 4692-4696(2002)). Thirdly, T1R1+3 is activated by psychophysically relevantconcentrations of the umami agonists L-Asp and L-AP4 (Nelson, G. et al.,Nature, 416, 199-202 (2002); Li, X. et al., Proc Natl Acad Sci USA, 99,4692-4696 (2002)). In order to rigorously assess the role of T1R1+3 inumami taste, we examined T1R1 and T1R3 KO animals (see FIG. 1).

Because of its Na+ content, monosodium glutamate (MSG) evokes both saltyand umami taste. We therefore assayed umami responses using severalstrategies that allowed us to isolate salt taste from that of glutamatein behavioral and electrophysiological studies. These included testingMSG in the presence of the sodium channel blocker amiloride, using MPG,the potassium salt of glutamate, and testing the umami agonists AP4 andaspartic acid, all in the presence or absence of the umami enhancer IMP.FIG. 3 shows that when salt effects are minimized, T1R3 KO mice have adramatic loss of behavioral attraction—and a profound correspondingdeficit in physiological responses to all umami tastants—includingglutamate, aspartate, glutamate plus IMP, and IMP alone. Very recently,Damak et al independently generated T1R3 KO animals but concluded thatmultiple umami receptors must exist as significant MSG responsesremained in their studies of KO mice (Damak, S. et al., Science, 301,850-853 (2003)). Notably, the MSG responses of the KO animals werestrictly independent of IMP, a contradiction given that IMP enhancementis the hallmark of the umami modality. Since salt effects were notaccounted for, we suspect that much of their remaining responses reflectNa+ content in MSG rather than umami taste (compare responses to MSG+IMPversus MPG+IMP or MSG+IMP+amiloride in FIG. 3 e-f).

If T1R1 combines with T1R3 (T1R1+3) to generate the mammalian umamireceptor, then a knockout of T1R1 should also eliminate all umamiresponses. FIG. 3 demonstrates that this is absolutely the case. Incontrast, these very same tastants elicit normal, robust responses incontrol and in T1R2 KO animals. Together, these results prove thatT1R1+3 is the mammalian umami receptor.

Previously, we showed that in addition to typical umami tastants, themouse T1R1+3 receptor is also activated by other L-amino amino acids,and in the presence of IMP functions as a broadly tuned L-amino acidsensor (Nelson, G. et al., Nature, 416, 199-202 (2002)). Therefore, wetested responses of T1R1 and T1R3 KO animals to L-amino acids in thepresence or absence of IMP. Indeed, responses to amino acid tastants areseverely defective in T1R1 and T1R3, but not T1R2 KO strains (FIG. 3),firmly establishing the T1R1+3 heteromeric GPCR complex as the tastereceptor for a wide range of L-amino acids and IMP. Interestingly, whenwe assayed exceedingly high concentrations of L-amino acids that tastesweet to humans (e.g. >300 mM Ala, Ser, and Thr), T1R1 KO animals, butnot T1R3 KO mice retained a small residual attraction (see panel d inFIG. 3); these trace behavioral responses likely reflect the activationof the T1R2+3 sweet taste receptor (Nelson, G. et al., Cell, 106,381-390 (2001); see below).

T1R2+3 and T1R3 are Required for Sweet Reception and Perception

T1R2+3 functions in cell based assays as a heteromeric receptor fordiverse chemical classes of sweet compounds including natural sugars,artificial sweeteners, Damino acids and sweet-tasting proteins (Nelson,G. et al., Cell, 106, 381-390 (2001); Li, X. et al., Proc Natl Acad SciUSA, 99, 4692-4696 (2002)). However, a number of studies have suggestedthat animals may express distinct types of sweet receptors (Schiffman,S. S. et al., Pharmacol Biochem Behav, 15, 377-388 (1981); Ninomiya, Y.et al., J Neurophysiol, 81, 3087-3091 (1999)). To define the role ofT1R2+3 in vivo, we examined sweet responses of knockout mice that lackfunctional T1R2 and T1R3 proteins. FIGS. 4 and 5 demonstrate thatresponses to all classes of sweet tastants are dramatically impaired inT1R2 and T1R3 knockout strains. We tested a broad panel of sugars,artificial sweeteners and D-amino acids, and in all cases responses wereseverely defective: behavioral attraction is nearly abolished and nerveresponses are greatly diminished. These results confirm T1R2+3 as theprincipal sweet taste sensor in vivo.

Notably, very high concentrations (>300 mM) of natural sugars, but notof artificial sweeteners or D-amino acids, elicited modest butdetectable attractive responses in both T1R2 and T1R3 knockout strains.Thus, either there are additional sweet taste receptors (i.e.T1R-independent pathways), or T1R2 and T1R3 may also function on theirown as low affinity receptors for natural sugars in the absence of theirheteromeric partners. If the remaining responses are in fact due to T1R2or T1R3, then a double knockout of these GPCRs should eliminate allsweet responses. Since T1R2 and T1R3 loci are linked at the distal endof chromosome 4 (Nelson, G. et al., Cell, 106, 381-390 (2001)), we firstgenerated recombinant T1R2 KO, T1R3 KO mice and then tested themphysiologically and behaviorally. FIGS. 4 and 5 (red traces) show thatT1R2, T1R3 double KO mice have lost all responses to high concentrationof sugars. Together, these results illustrate the in vivo significanceof the combinatorial assembly of T1Rs, and demonstrate that all sweettaste reception operates via the T1R2 and T1R3 GPCRs.

Do T1R2 or T1R3 homodimeric receptors play a significant role in sweetsensing in wild type mice? T1R2 is always expressed in cells containingT1R3 (T1R2+3 cells; Nelson, G. et al., Cell, 106, 381-390 (2001)).Therefore, even if some T1R2 were not associated with T1R3 in thesecells, the much higher affinity of the T1R2+3 heteromeric receptor forsweet tastants would likely dominate the cellular response. In contrast,we previously reported that T1R3 is also found in a significant fractionof cells of the tongue and palate epithelium independent of T1R1 andT1R2 (T1R3 alone cells; Nelson, G. et al., Cell, 106, 381-390 (2001)).This class of cells may provide animals with additional means ofdetecting and responding to high concentrations of sugars. Todemonstrate that T1R3 alone can function as a low affinity receptor fornatural sugars, we generated HEK cells stably expressing T1R3 and anoptimized G protein chimera engineered to couple to T1Rs (seeExperimental Procedures). FIG. 6 shows that T1R3 alone in fact respondsto very high concentrations of natural sugars, but not to lowerconcentrations (<300 mM), or to artificial sweeteners. These resultsconfirm T1R3 as a low affinity sugar receptor, and support the postulatethat T1R3 alone cells function in vivo as additional sweet sensors(Nelson, G. et al., Cell, 106, 381-390 (2001)). This partial cellularsegregation of sensing natural and artificial sweeteners may helpexplain why artificial sweeteners never attain the level of sweetnessafforded by high concentrations of natural sugars (i.e. activation ofT1R2+3 cells versus T1R2+3 and T1R3 alone cells).

T1R2 Delimits Species-Specific Sweet Taste Preferences

Humans can taste a number of natural and artificial sweeteners thatrodents cannot. For example, monellin, thaumatin, aspartame andneohesperidin dihydrochalcone taste sweet to humans at sub-millimolarconcentrations, whereas rodents show no preference even at 100 timeshigher concentrations (Danilova, V. et al., J Neurophysiol, 80,2102-2112 (1998)). Previously, we reported that rodent and human T1Rsare more than 30% dissimilar in their amino acid sequences, andhypothesized that such differences underlie the species-specificselectivity in sweet taste detection (Nelson, G. et al., Cell, 106,381-390 (2001); Nelson, G. et al., Nature, 416, 199-202 (2002)). BecauseT1R2 participates exclusively in sweet taste detection while T1R3 isinvolved in both sweet and amino acid recognition, we reasoned that T1R2would be a particularly critical determinant of sweet taste selectivityin vivo. Therefore, we predicted that introducing the human T1R2 gene inT1R2 KO mice should both rescue and “humanize” sweet responses.

We generated mice that were homozygous for the T1R2 KO allele, butinstead expressed a human T1R2 transgene in the native “T1R2-cells”. A12 kb genomic clone containing the T1R2 regulatory sequences was fusedto a hT1R2 full length cDNA and introduced into T1R2 KO mice. Multipleindependent lines were assayed for their selectivity and sensitivity tosweet tastants. To examine expression of hT1R2, we performed two-colorfluorescent in situ hybridization experiments in transgenic animalscarrying the wild type mT1R2 allele. FIG. 7 (panel a-d) demonstrate thathuman T1R2 is selectively expressed in T1R2-expressing cells, andeffectively restores sweet taste function. More importantly, the humantransgene now confers these mice with the ability to detect and respondto several compounds that taste sweet to humans, but are not normallyattractive to rodents; these include aspartame, glycyrrhizic acid andthe sweet proteins thaumatin and monellin. Interestingly, the humanizedT1R2 mice still do not respond to the intensely sweet compoundneohesperidin dihydrochalcone, nor do HEK cells transfected with thehuman T1R2 and mouse T1R3 GPCRs. However, when cells are transfectedwith human T1R2 and human T1R3 they robustly respond to neohesperidindihydrochalcone. Taken together, these experiments validate T1Rs as keydeterminants of differences in sweet taste selectivity and specificitybetween rodents and humans, and further substantiate T1R2+3, andT1R2-expressing cells, as an principal mediator of sweet taste in vivo.Finally, we propose that polymorphisms in both T1R2 and T1R3 areimportant determinants of human individual sweet taste preferences.

T1R2-Expressing Cells Encode Behavioral Attraction in vivo

Activation of taste receptors trigger distinct behavioral responses inanimals. For example, excitation of the T1R2+3 receptor stimulatesbehavioral attraction to sugars and sweet-tasting compounds in mice. Isthis response a property of the receptors or the cells in which they areexpressed? One way to answer this question would be to express a novelreceptor unrelated to the taste system in the T1R2+3 cells and examinewhether its selective stimulation elicits attractive responses (Troemel,E. R. et al., Cell 91, 161-169 (1997)).

Our approach was to target expression of a GPCR that could couple to theendogenous signaling pathways in T1R2+3 cells, but could only beactivated by a nonnatural ligand. In order to examine taste responses inthe very same animals before and after receptor expression we utilizedan inducible system. To accomplish this, we used transgenic mice inwhich a modified k-opioid receptor activated solely by a syntheticligand (RASSL; Redfern, C. H. et al., Nat Biotechnol, 17, 165-169(1999)) was targeted to the T1R2-expressing cells under the control ofthe Tet-on inducible system (see Experimental Procedures).

FIG. 7 e shows that un-induced animals, or wild type controls treatedwith doxycycline, are completely insensitive to the k-opioid agonistspiradoline. Remarkably, induction of RASSL expression in the T1R2-cellsgenerates animals that are now strongly attracted to nanomolarconcentrations of spiradoline (FIG. 7, red trace). Thus, we concludethat activation of T1R2-expressing cells, rather than the receptors theyexpress, determines behavioral attraction in mice. Furthermore, theseresults unequivocally show that activating a single cell type issufficient to trigger specific taste responses; therefore a modelrequiring a combinatorial pattern of activity, or temporal coding, isnot needed to account for attraction mediated by T1R2-expressing cells.By extension we suggest that activation of these taste signalingpathways in human T1R2+3 cells, regardless of the nature of thereceptor, would evoke sweet taste.

Multiple receptors have been proposed to mediate sweet and umami tastein mammals. Notably, even within each of these two modalities severalGPCRs, ion channels, and models invoking intracellular targets directlyactivated by cell-permeable tastants have been postulated (Kinnamon, S.C. Neuron, 25, 507-510 (2000); Margolskee, R. F., J Biol Chem, 277, 1-4(2002)). We have used a combination of cell-based assays, genetic,physiological and behavioral approaches to prove that the receptors forsweet and umami taste in mammals are the T1Rs: umami taste is mediatedby the T1R1+3 heteromeric GPCR, and sweet by the two T1R-basedreceptors, T1R2 and T1R3 (T1R2+3, and most likely, a homodimer of T1R3).Therefore, sweet and amino acid taste (umami)-two chemosensory inputsthat trigger behavioral attraction, share a common receptor repertoireand evolutionary origin.

The human T1R1+3 receptor is activated by glutamate and aspartate farmore effectively than by other amino acids (Li, X. et al., Proc NatlAcad Sci USA, 99, 4692-4696 (2002)). In contrast, the mouse T1R1+3receptor recognizes a much broader range of L-amino acids, both in cellbased assays (Nelson, G. et al., Nature, 416, 199-202 (2002)) and invivo (this paper). If the evolutionary role of the T1R1+3 receptor wasto mediate attractive responses to protein-rich foods, one may questionwhether the tuning of receptor selectivity in primates to just two aminoacids substantially altered the ability to detect diets rich thesenutrients. Since amino acids are usually found as complex mixtures,detecting any one should generally be adequate, and thus this“narrowing” of tastant selectivity should not have had a significantdietary impact. Given that the same cells and receptors recognizeglutamate, other amino acids and IMP, we suggest that in rodents theumami taste modality must be generalized to include most L-amino acidsand the very concept of a distinct glutamate taste in rodents(Chaudhari, N. et al., Nat Neurosci, 3, 113-119 (2000); Lin, W. et al.,J Neurophysiol, 89, 1434-1439 (2003)) needs to be re-evaluated.

A spoonful of sugar or a few tablets of artificial sweetener? Our day today experiences tell us that natural and artificial sweeteners do nottaste the same. In this manuscript we showed that T1R2 and T1R3 areresponsible for all sweet sensing. How do they account for the perceivedtaste differences between sweet tastants? Many sweeteners are likely toactivate receptors for other taste modalities, like T2R bitter sensingcells accounting for the bitter aftertaste of saccharin (data notshown). Therefore, the “taste” of even a single sweet molecular speciesmay reflect the combined activity of cells tuned to different tastemodalities, and not just the activity of sweet sensing cells. We havealso shown that at higher, but still physiologically relevantconcentrations of sugars (>300 mM), natural and artificial sweetenersactivate partially overlapping, yet distinct sweet receptor types(T1R2+3 and T1R3 alone).

We have shown that T1Rs are the mediators of the two principalattractive taste modalities, and demonstrated that mice expressing aRASSL opioid receptor became powerfully attracted to spiradoline, anormally tasteless and nutrionally irrelevant compound, proving that totaste is to believe. The discovery and functional characterization ofthe cells and receptors for bitter, sweet, and umami taste now provide acompelling view of how taste is encoded at the periphery: dedicatedtaste receptor cells mediate attractive and aversive behaviors (see,e.g., Zhang, Y. et al., Cell, 112, 293-301 (2003)).

EXPERIMENTAL PROCEDURES

Gene Targeting of T1R1, T1R2 and T1R3

The strategy used to create T1R knockout animals is shown in FIG. 1. ForT1R1, exon 6 encoding the predicted seven transmembrane domain of thereceptor was replaced by the PGK-neo^(r) cassette. Homologousrecombination in R1 ES cells was detected by diagnostic Southernhybridization with probes outside the targeting construct. Two targetedES clones were injected into C57BL/6 blastocysts. Chimeric mice werebred with C57BL/6 mice and progeny backcrossed to C57BL/6 mice for twogenerations prior to establishing a homozygous knockout colony.

For T1R2, a similar approach deleted exons 5 and 6 (see FIG. 1).Chimeric animals were bred with C57BL/6 mice and progeny backcrossed toC57BL/6 mice for four generations. The T1R3 taster(C57) and non-taster(129) alleles (Nelson, G. et al., Cell, 106, 381-390 (2001)) wereidentified based on an EcoRI polymorphism ˜12 kb upstream of thestarting ATG of T1R3. All of the T1R2 knockout animals used in thisstudy carried a taster allele of T1R3. However, studies with T1R2 KOmice homozygous for the non-taster T1R3 allele produced qualitativelysimilar results (data not shown). To generate T1R3 KO knockout animals,we replaced exons 1 to 5 encoding the N-terminal extracellular domainwith the PGK-neor cassette (see FIG. 1). Chimeric mice were bred withC57BL/6 mice and progeny backcrossed to C57BL/6 mice for twogenerations.

T1R knockouts have normal viability, body weight, overall anatomy andgeneral behavior. Similarly, taste receptor cells appear normalmorphologically and numerically in all knockout backgrounds.

In Situ Hybridization

Fresh frozen sections (16 μm/section) were attached to silanized slidesand prepared for in situ hybridization or immunohistochemistry aspreviously described (Hoon, M. A. et al., Cell., 96, 541-551 (1999)).Single label in situ hybridization was carried out using digoxigeninlabeled probes; T1R1 and T1R2 probes were to the predicted transmembranedomains, while T1R3 and RASSL (Redfern, C. H. et al., Nat Biotechnol,17, 165-169 (1999)) probes utilized the full coding sequences.Double-label fluorescent detection used fluorescein (full-length hT1R2)and digoxigenin (full-length mT1R2) probes at high stringency(hybridization, 5× SSC, 50% formamide, 65-72° C.; washing, 0.2× SSC, 72°C.). Hybridization was detected with distinct fluorescent substrates(Adler, E. et al., Cell, 100, 693-702 (2000)) and specificity oflabeling was checked using T1R2-knockout and non transgenic controls.

Generation of Transgenic Mice Expressing Human T1R2 and RASSL

An approx. 12 kb genomic fragment upstream of mouse T1R2 was fused to ahuman T1R2 cDNA and to a reverse-tetracycline dependent transactivator(rtTA) construct (Gossen, M. et al., Curr Opin Biotechnol, 5, 516-520(1994)). Transgenic lines were produced by pronuclear injection ofzygotes from FVB/N mice. Three independent human T1R2 transgenic linesdisplayed behavioral attraction to aspartame (10 mM). One line wascrossed into the T1R2 knockout background, and assayed for tasteresponses and transgene expression. No expression outside T1R2-cells wasdetected. T1R2-rtTA transgenic lines were crossed withtetO-Ro1/tetO-lacZ transgenic animals (Redfern, C. H. et al., NatBiotechnol, 17, 165-169 (1999)). Doubleheterozygous progeny were inducedby doxycycline treatment (6 gm/kg) (Bio-Serv) for 3 days (Gogos, J. A.et al., Cell, 103, 609-620 (2000)) and examined for β-galactosidaseactivity (Zack, D. J. et al., Neuron 6, 187-199 (1991)) and RASSLexpression in the tongue and palate. A line displaying appropriateβ-galactosidase staining and RASSL expression pattern was selected forbehavioral assays.

Behavioral Assays

Taste behavior was assayed using a short term assay that directlymeasures taste preferences by counting immediate licking responses in amulti-channel gustometer (Davis MS160-Mouse gustometer; DiLogInstruments, Tallahassee, Fla.). Mice were trained and tested asdescribed previously (Zhang, Y. et al., Cell, 112, 293-301 (2003)).Individual mice were placed in the gustometer for 30 minutes, andstimuli were presented in random order for 5 s trials that wereinitiated by the mouse licking the stimulus spout. For sodium saccharin,glutamate and aspartate, 100 μM amiloride was added to all solutions(including the control) to minimize effects of salt taste. Data pointsrepresent the mean rate that mice licked a tastant relative to theirsampling of an appropriate control tastant (ratio defined as lick raterelative to control); lick suppression is defined as 1 minus the lickrate relative to control. In most cases the control tastant was waterbut for amino acids +1 mM IMP, 200 mM MSG and 10 mM IMP the controlswere 1 mM IMP, 200 mM sodium gluconate and 10 mM CMP, respectively.

Standard two-bottle preference assays were carried out as describedpreviously (Nelson, G. et al., Cell, 106, 381-390 (2001)). For micecarrying T1R2-rtTA and tetO-Ro1/tetO-lacZ transgenes, expression wasinduced by doxycycline treatment 3 days prior to, and during thebehavioral testing. Controls included testing the same mice withoutinduction as well as mice carrying just the T1R2-rtTA transgene treatedwith doxycycline. All three groups displayed normal responses tosucrose.

We noted that in 2-bottle assays T1R-KO animals appear to “learn” toidentify solutions containing very high concentrations of natural sugars(>500 mM); successive exposure resulted in decreased detection thresholdand increased preference ratios. Because mice are repeatedly exposed totest compounds for 48 hrs in standard twobottle assays, they may useother sensory inputs like texture or smell to distinguish tastant fromwater. If not properly controlled, this could be easily misunderstood asbehavioral attraction via taste pathways. To avoid this problem, we usedeither short term immediate lick response assays (see above) ortwo-bottle assays with naive knockout mice (i.e. never exposed to suchtastants during either training or testing).

Nerve Recordings

Lingual stimulation and recording procedures were performed aspreviously described (Dahl, M. et al., Brain Res, 756, 22-34 (1997);Nelson, G. et al., Nature, 416, 199-202 (2002)). Neural signals wereamplified (5,000×) with a Grass P511 AC amplifier (Astro-Med), digitizedwith a Digidata 1200B A/D converter (Axon Instruments), and integrated(r.m.s. voltage) with a time constant of 0.5 s. Taste stimuli werepresented at a constant flow rate of 4 ml min⁻¹ for 20 s intervalsinterspersed by 2 min rinses with artificial saliva (Danilova, V., andHellekant, G., BMC Neurosci, 4, 5. (2003)) between presentations. Alldata analyses used the integrated response over a 25 s periodimmediately after the application of the stimulus. Each experimentalseries consisted of the application of 6 tastants bracketed bypresentations of 0.1 M citric acid to ensure the stability of therecording. The mean response to 0.1 M citric acid was used to normalizeresponses to each experimental series.

Tastants used for nerve recordings (maximal concentrations) were:sucrose, glucose, maltose (600 mM); sodium saccharin (40 mM); AceK (60mM); Citric Acid (100 mM); NaCl (100 mM); NH₄Cl (100 mM); 6-n-propylthiouracil (10 mM), quinine (10 mM); cycloheximide (1 mM); L-Ser, L-Ala,(30 mM with 0.5 mM IMP added) MSG and MPG (300 mM with or without 0.5 mMIMP); D-Ala, D-Phe, and D-Trp (100 mM). Amiloride (50 uM) was added toreduce sodium responses as indicated in the figure legends.

Heterologous Expression of T1Rs and Calcium Imaging

Modified HEK-293 cells (PEAK^(rapid) cells; Edge BioSystems, Md.) weregrown, transfected with T1Rs and promiscuous G-proteins and assayed forfunctional responses to tastants by Ca-imaging essentially as describedpreviously (Nelson, G. et al., Cell, 106, 381-390 (2001)). Minordifferences in FURA-2 loading and Ca-imaging included using 199(H)Medium (Biosource) containing 0.1% BSA, 100 μM EGTA and 200 μM CaCl₂ asassay buffer as well as reducing the time allowed for FURA-AM estercleavage to 10 minutes. The imaging system was an Olympus IX50microscope equipped with a 10×/0.5 N.A. fluor objective (Zeiss), theTILL imaging system (TILL Photonics GmbH), and a cooled CCD camera.Acquisition and analysis of fluorescence images used TILL-Visionsoftware.

To optimize coupling of T1R-responses to changes in [Ca²⁺]i, C-terminalresidues of human Gα16 (Offermanns, S., and Simon, M. I., J Biol Chem,270, 15175-15180 (1995)) were replaced with the corresponding residuesfrom Gz (Mody, S. M. et al., Mol Pharmacol, 57, 13-23 (2000)), gustducin(McLaughlin, S. K. et al., Nature, 357, 563-569 (1992)) or Gαi2. Achimera containing the C-terminal 25 residues of gustducin (G_(gust-25))proved particularly effective at mediating responses of mouse T1R2+3 andT1R1+3 in transient transfection assays, and was used for furtherstudies. Cell lines stably expressing T1R3 and G_(gust-25) wereestablished using puromycin and Zeocin (Invitrogen) selection. Threeindependent lines expressing T1R3 and G_(gust-25) were used to examinethe specificity and dose response of the T1R3 receptor. Sucrose andmaltose (>300 mM) elicited dose dependent responses that were T1R3 andG_(gust-25) dependent, but attempts to use high concentrations ofseveral other sugars (glucose, fructose, trehalose and galactose) provedimpractical because they induced significant receptor independent risesin [Ca²⁺]i.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Informal Sequence Listing

T1R1 SEQUENCES Rat T1R1 amino acid sequence--SEQ ID NO:1MLFWAAHLLLSLQLVYCWAFSCQRTESSPGFSLPGDFLLAGLFSLHGDCLQVRHRPLVTSCDRPDSFNGHGYHLFQAMRFTVEEINNSSALLPNITLGYELYDVCSESANVYATLRVLALQGPRHIEIQKDLRNHSSKVVAFIGPDNTDHAVTTAALLGPFLMPLVSYEASSVVLSAKRKFPSFLRTVPSDRHQVEVMVQLLQSFGWVWISLIGSYGDYGQLGVQALEELAVPRGICVAFKDIVPFSARVGDPRMQSMMQHLAQARTTVVVVFSNRHLARVFFRSVVLANLTGKVWVASEDWAISTYITSVTGIQGIGTVLGVAVQQRQVPGLKEFEESYVRAVTAAPSACPEGSWCSTNQLCRECHTFTTRNMPTLGAFSMSAAYRVYEAVYAVAHGLHQLLGCTSEICSRGPVYPWQLLQQIYKVNFLLHENTVAFDDNGDTLGYYDIIAWDWNGPEWTFEIIGSASLSPVHLDINKTKIQWHGKNNQVPVSVCTTDCLAGHHRVVVGSHHCCFECVPCEAGTFLNMSELHICQPCGTEEWAPKESTTCFPRTVEFLAWHEPISLVLIAANTLLLLLLVGTAGLFAWHFHTPVVRSAGGRLCFLMLGSLVAGSCSFYSFFGEPTVPACLLRQPLFSLGFAIFLSCLTIRSFQLVIIFKFSTKVPTFYRTWAQNHGAGLFVIVSSTVHLLICLTWLVMWTPRPTREYQRFPHLVILECTEVNSVGFLLAFTHNILLSISTFVCSYLGKELPENYNEAKCVTFSLLLNFVSWIAFFTMASIYQGSYLPAVNVLAGLTTLSGGFSGYFLPKCYVILCRPELNNTEHFQASIQDYTRRCGTT Mouse T1R1 amino acid sequence--SEQID NO:2 MLFWAAHLLLSLQLAVAYCWAFSCQRTESSPGFSLPGDFLLAGLFSLHADCLQVRHRPLVTSCDRSDSFNGHGYHLFQAMRFTVEEINNSTALLPNITLGYELYDVCSESSNVYATLRVPAQQGTGHLEMQRDLRNHSSKVVALIGPDNTDHAVTTAALLSPFLMPLVSYEASSVILSGKRKFPSFLRTIPSDKYQVEVIVRLLQSFGWVWISLVGSYGDYGQLGVQALEELATPRGICVAFKDVVPLSAQAGDPRMQRMMLRLARARTTVVVVFSNRHLAGVFFRSVVLANLTGKVWIASEDWAISTYITNVPGIQGIGTVLGVAIQQRQVPGLKEFEESYVQAVMGAPRTCPEGSWCGTNQLCRECHAFTTWNMPELGAFSMSAAYNVYEAVYAVAHGLHQLLGCTSGTCARGPVYPWQLLQQIYKVNFLLHKKTVAFDDKGDPLGYYDIIAWDWNGPEWTFEVIGSASLSPVHLDINKTKIQWHGKNNQVPVSVCTRDCLEGHHRLVMGSHHCCFECMPCEAGTFLNTSELHTCQPCGTEEWAPEGSSACFSRTVEFLGWHEPISLVLLAANTLLLLLLIGTAGLFAWRLHTPVVRSAGGRLCFLMLGSLVAGSCSLYSFFGKPTVPACLLRQPLFSLGFAIFLSCLTIRSFQLVIIFKFSTKVPTFYHTWAQNHGAGIFVIVSSTVHLFLCLTWLAMWTPRPTREYQRFPHLVILECTEVNSVGFLVAFAHNILLSISTFVCSYLGKELPENYNEAKCVTFSLLLHFVSWIAFFTMSSIYQGSYLPAVNVLAGLATLSGGFSGYFLPKCYVILCRPELNNTEHFQASIQDYTRRCGTT Human T1R1 amino acid sequence--SEQID NO:3 MLLCTARLVGLQLLISCCWAFACHSTESSPDFTLPGDYLLAGLFPLHSGCLQVRHRPEVTLCDRSCSFNEHGYHLFQAMRLGVEEINNSTALLPNITLGYQLYDVCSDSANVYATLRVLSLPGQHHIELQGDLLHYSPTVLAVIGPDSTNRAATTAALLSPFLVHISYAASSETLSVKRQYPSFLRTIPNDKYQVETMVLLLQKFGWTWISLVGSSDDYGQLGVQALENQALVRGICIAFKDIMPFSAQVGDERMQCLMRHLAQAGATVVVVFSSRQLARVFFESVVLTNLTGKVWVASEAWALSRHITGVPGIQRIGMVLGVAIQKRAVPGLKAFEEAYARADKEAPRPCHKGSWCSSNQLCRECQAFMAHTMPKLKAFSMSSAYNAYRAVYAVAHGLHQLLGCASELCSRGRVYPWQLLEQIHKVHFLLHKDTVAFNDNRDPLSSYNIIAWDWNGPKWTFTVLGSSTWSPVQLNINETKIQWHGKNHQVPKSVCSSDCLEGHQRVVTGFHHCCFECVPCCAGTFLNKSELYRCQPCGTEEWAPEGSQTCFPRTVVFLALREHTSWVLLAANTLLLLLLLGTAGLFAWHLDTPVVRSAGGRLCFLMLGSLAAGSGSLYGFFGEPTRPACLLRQALFALGFTIFLSCLTVRSFQLIIIFKFSTKVPTFYHAWVQNHGAGLFVMISSAAQLLICLTWLVVWTPLPAREYQRFPHLVMLECTETNSLGFILAFLYNGLLSISAFACSYLGKDLPENYNEAKCVTFSLLFNFVSWIAFFTTASVYDGKYLPAANMMAGLSSLSSGFGGYFLPKCYVILCRPDLNSTEHFQASIQDYTRRCGST Rat T1R1 nucleotide sequence--SEQ IDNO:4 ATTCACATCAGAGCTGTGCTCAGCCATGCTGGGCAGAGGGACGACGGCTGGCCAGCATGCTCTTCTGGGCTGCTCACCTGCTGCTCAGCCTGCAGTTGGTCTACTGCTGGGCTTTCAGCTGCCAAAGGACAGAGTCCTCTCCAGGCTTCAGCCTTCCTGGGGACTTCCTCCTTGCAGGTCTGTTCTCCCTCCATGGTGACTGTCTGCAGGTGAGACACAGACCTCTGGTGACAAGTTGTGACAGGCCCGACAGCTTCAACGGCCATGGCTACCACCTCTTCCAAGCCATGCGGTTCACTGTTGAGGAGATAAACAACTCCTCGGCCCTGCTTCCCAACATCACCCTGGGGTATGAGCTGTACGACGTGTGCTCAGAATCTGCCAATGTGTATGCCACCCTGAGGGTGCTTGCCCTGCAAGGGCCCCGCCACATAGAGATACAGAAAGACCTTCGCAACCACTCCTCCAAGGTGGTGGCCTTCATCGGGCCTGACAACACTGACCACGCTGTCACTACCGCTGCCTTGCTGGGTCCTTTCCTGATGCCCCTGGTCAGCTATGAGGCAAGCAGCGTGGTACTCAGTGCCAAGCGCAAGTTCCCGTCTTTCCTTCGTACCGTCCCCAGTGACCGGCACCAGGTGGAGGTCATGGTGCAGCTGCTGCAGAGTTTTGGGTGGGTGTGGATCTCGCTCATTGGCAGCTACGGTGATTACGGGCAGCTGGGTGTGCAGGCGCTGGAGGAGCTGGCCGTGCCCCGGGGCATCTGCGTCGCCTTCAAGGACATCGTGCCTTTCTCTGCCCGGGTGGGTGACCCGAGGATGCAGAGCATGATGCAGCATCTGGCTCAGGCCAGGACCACCGTGGTTGTGGTCTTCTCTAACCGGCACCTGGCTAGAGTGTTCTTCAGGTCCGTGGTGCTGGCCAACCTGACTGGCAAAGTGTGGGTCGCCTCAGAAGACTGGGCCATCTCCACGTACATCACCAGCGTGACTGGGATCCAAGGCATTGGGACGGTGCTCGGTGTGGCCGTCCAGCAGAGACAAGTCCCTGGGCTGAAGGAGTTTGAGGAGTCTTATGTCAGGGCTGTAACAGCTGCTCCCAGCGCTTGCCCGGAGGGGTCCTGGTGCAGCACTAACCAGCTGTGCCGGGAGTGCCACACGTTCACGACTCGTAACATGCCCACGCTTGGAGCCTTCTCCATGAGTGCCGCCTACAGAGTGTATGAGGCTGTGTACGCTGTGGCCCACGGCCTCCACCAGCTCCTGGGATGTACTTCTGAGATCTGTTCCAGAGGCCCAGTCTACCCCTGGCAGCTTCTTCAGCAGATCTACAAGGTGAATTTTCTTCTACATGAGAATACTGTGGCATTTGATGACAACGGGGACACTCTAGGTTACTACGACATCATCGCCTGGGACTGGAATGGACCTGAATGGACCTTTGAGATCATTGGCTCTGCCTCACTGTCTCCAGTTCATCTGGACATAAATAAGACAAAAATCCAGTGGCACGGGAAGAACAATCAGGTGCCTGTGTCAGTGTGTACCACGGACTGTCTGGCAGGGCACCACAGGGTGGTTGTGGGTTCCCACCACTGCTGCTTTGAGTGTGTGCCCTGCGAAGCTGGGACCTTTCTCAACATGAGTGAGCTTCACATCTGCCAGCCTTGTGGAACAGAAGAATGGGCACCCAAGGAGAGCACTACTTGCTTCCCACGCACGGTGGAGTTCTTGGCTTGGCATGAACCCATCTCTTTGGTGCTAATAGCAGCTAACACGCTATTGCTGCTGCTGCTGGTTGGGACTGCTGGCCTGTTTGCCTGGCATTTTCACACACCTGTAGTGAGGTCAGCTGGGGGTAGGCTGTGCTTCCTCATGCTGGGTTCCCTGGTGGCCGGAAGTTGCAGCTTCTATAGCTTCTTCGGGGAGCCCACGGTGCCCGCGTGCTTGCTGCGTCAGCCCCTCTTTTCTCTCGGGTTTGCCATCTTCCTCTCCTGCCTGACAATCCGCTCCTTCCAACTGGTCATCATCTTCAAGTTTTCTACCAAGGTGCCCACATTCTACCGTACCTGGGCCCAAAACCATGGTGCAGGTCTATTCGTCATTGTCAGCTCCACGGTCCATTTGCTCATCTGTCTCACATGGCTTGTAATGTGGACCCCACGACCCACCAGGGAATACCAGCGCTTCCCCCATCTGGTGATTCTCGAGTGCACAGAGGTCAACTCTGTAGGCTTCCTGTTGGCTTTCACCCACAACATTCTCCTCTCCATCAGTACCTTCGTCTGCAGCTACCTGGGTAAGGAACTGCCAGAGAACTATAATGAAGCCAAATGTGTCACCTTCAGCCTGCTCCTCAACTTCGTATCCTGGATCGCCTTCTTCACCATGGCCAGCATTTACCAGGGCAGCTACCTGCCTGCGGTCAATGTGCTGGCAGGGCTGACCACACTGAGCGGCGGCTTCAGCGGTTACTTCCTCCCCAAGTGCTATGTGATTCTCTGCCGTCCAGAACTCAACAATACAGAACACTTTCAGGCCTCCATCCAGGACTACACGAGGCGCTGCGGCACTACCTGATCCACTGGAAAGGTGCAGACGGGAAGGAAGCCTCTCTTCTTGTGCTGAAGGTGGCGGGTCCAGTGGGGCCGAGAGCTTGAGGTGTCTGGGAGAGCTCCGGCACAGCTTACGATGTATAAGCACGCGGAAGAATCCAGTGCAATAAAGACGGGAAGTGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Mouse T1R1 nucleotidesequence--SEQ ID NO:5TTTGGCCAGCATGCTTTTCTGGGCAGCTCACCTGCTGCTCAGCCTGCAGCTGGCCGTTGCTTACTGCTGGGCTTTCAGCTGCCAAAGGACAGAATCCTCTCCAGGTTTCAGCCTCCCTGGGGACTTCCTCCTGGCAGGCCTGTTCTCCCTCCATGCTGACTGTCTGCAGGTGAGACACAGACCTCTGGTGACAAGTTGTGACAGGTCTGACAGCTTCAACGGCCATGGCTATCACCTCTTCCAAGCCATGCGGTTCACCGTTGAGGAGATAAACAACTCCACAGCTCTGCTTCCCAACATCACCCTGGGGTATGAACTGTATGACGTGTGCTCAGAGTCTTCCAATGTCTATGCCACCCTGAGGGTGCCCGCCCAGCAAGGGACAGGCCACCTAGAGATGCAGAGAGATCTTCGCAACCACTCCTCCAAGGTGGTGGCACTCATTGGGCCTGATAACACTGACCACGCTGTCACCACTGCTGCCCTGCTGAGCCCTTTTCTGATGCCCCTGGTCAGCTATGAGGCGAGCAGCGTGATCCTCAGTGGGAAGCGCAAGTTCCCGTCCTTCTTGCGCACCATCCCCAGCGATAAGTACCAGGTGGAAGTCATAGTGCGGCTGCTGCAGAGCTTCGGCTGGGTCTGGATCTCGCTCGTTGGCAGCTATGGTGACTACGGGCAGCTGGGCGTACAGGCGCTGGAGGAGCTGGCCACTCCACGGGGCATCTGCGTCGCCTTCAAGGACGTGGTGCCTCTCTCCGCCCAGGCGGGTGACCCAAGGATGCAGCGCATGATGCTGCGTCTGGCTCGAGCCAGGACCACCGTGGTCGTGGTCTTCTCTAACCGGCACCTGGCTGGAGTGTTCTTCAGGTCTGTGGTGCTGGCCAACCTGACTGGCAAAGTGTGGATCGCCTCCGAAGACTGGGCCATCTCCACGTACATCACCAATGTGCCCGGGATCCAGGGCATTGGGACGGTGCTGGGGGTGGCCATCCAGCAGAGACAAGTCCCTGGCCTGAAGGAGTTTGAAGAGTCCTATGTCCAGGCAGTGATGGGTGCTCCCAGAACTTGCCCAGAGGGGTCCTGGTGCGGCACTAACCAGCTGTGCAGGGAGTGTCACGCTTTCACGACATGGAACATGCCCGAGCTTGGAGCCTTCTCCATGAGCGCTGCCTACAATGTGTATGAGGCTGTGTATGCTGTGGCCCACGGCCTCCACCAGCTCCTGGGATGTACCTCTGGGACCTGTGCCAGAGGCCCAGTCTACCCCTGGCAGCTTCTTCAGCAGATCTACAAGGTGAATTTCCTTCTACATAAGAAGACTGTAGCATTCGATGACAAGGGGGACCCTCTAGGTTATTATGACATCATCGCCTGGGACTGGAATGGACCTGAATGGACCTTTGAGGTCATTGGTTCTGCCTCACTGTCTCCAGTTCATCTAGACATAAATAAGACAAAAATCCAGTGGCACGGGAAGAACAATCAGGTGCCTGTGTCAGTGTGTACCAGGGACTGTCTCGAAGGGCACCACAGGTTGGTCATGGGTTCCCACCACTGCTGCTTCGAGTGCATGCCCTGTGAAGCTGGGACATTTCTCAACACGAGTGAGCTTCACACCTGCCAGCCTTGTGGAACAGAAGAATGGCCCCCTGAGGGGAGCTCAGCCTGCTTCTCACGCACCGTGGAGTTCTTGGGGTGGCATGAACCCATCTCTTTGGTGCTATTAGCAGCTAACACGCTATTGCTGCTGCTGCTGATTGGGACTGCTGGCCTGTTTGCCTGGCGTCTTCACACGCCTGTTGTGAGGTCAGCTGGGGCTAGGCTGTGCTTCCTCATGCTGGGTTCCTTGGTAGCTGGGAGTTGCAGCCTCTACAGCTTCTTCGGGAAGCCCACGGTGCCCGCGTGCTTGCTGCGTCAGCCCCTCTTTTCTCTCGGGTTTGCCATTTTCCTCTCCTGTCTGACAATCCGCTCCTTCCAACTGGTCATCATCTTCAAGTTTTCTACCAAGGTACCCACATTCTACCACACTTGGGCCCAAAACCATGGTGCCGGAATATTCGTCATTGTCAGCTCCACGGTCCATTTGTTCCTCTGTCTCACGTGGCTTGCAATGTGGACCCCACGGCCCACCAGGGAGTACCAGCGCTTCCCCCATCTGGTGATTCTTGAGTGCACAGAGGTCAACTCTGTGGGCTTCCTGGTGGCTTTCGCACACAACATCCTCCTCTCCATCAGCACCTTTGTCTGCAGCTACCTGGGTAAGGAACTGCCGGAGAACTATAACGAAGCCAAATGTGTCACCTTCAGCCTGCTCCTCCACTTCGTATCCTGGATCGCTTTCTTCACCATGTCCAGCATTTACCAGGGCAGCTACCTACCCGCGGTCAATGTGCTGGCAGGGCTGGCCACTCTGAGTGGCGGCTTCAGCGGCTATTTCCTCCCTAAATGCTACGTGATTCTCTGCCGTCCAGAACTCAACAACACAGAACACTTTCAGGCCTCCATCCAGGACTACACGAGGCGCTGCGGCACTACCTGAGGCGCTGCGGCACTACCTGAGGCGCTGCGGCACTACCTGA Human T1R1 nucleotidesequence--SEQ ID NO:6AGGTCTTGTAGCTTCAATGAGCATGGCTACCACCTCTTCCAGGCTATGCGGCTTGGGGTTGAGGAGATAAACAACTCCACGGCCCTGCTGCCCAACATCACCCTGGGGTACCAGCTGTATGATGTGTGTTCTGACTCTGCCAATGTGTATGCCACGCTGAGAGTGCTCTCCCTGCCAGGGCAACACCACATAGAGCTCCAAGGAGACCTTCTCCACTATTCCCCTACGGTGCTGGCAGTGATTGGGCCTGACAGCACCAACCGTGCTGCCACCACAGCCGCCCTGCTGAGCCCTTTCCTGGTGCATATTAGCTATGCGGCCAGCAGCGAGACGCTCAGCGTGAAGCGGCAGTATCCCTCTTTCCTGCGCACCATCCCCAATGACAAGTACCAGGTGGAGACCATGGTGCTGCTGCTGCAGAAGTTCGGGTGGACCTGGATCTCTCTGGTTGGCAGCAGTGACGACTATGGGCAGCTAGGGGTGCAGGCACTGGAGAACCAGGCCCTGGTCAGGGGCATCTGCATTGCTTTCAAGGACATCATGCCCTTCTCTGCCCAGGTGGGCGATGAGAGGATGCAGTGCCTCATGCGCCACCTGGCCCAGGCCGGGGCCACCGTCGTGGTTGTTTTTTCCAGCCGGCAGTTGGCCAGGGTGTTTTTCGAGTCCGTGGTGCTGACCAACCTGACTGGCAAGGTGTGGGTCGCCTCAGAAGCCTGGGCCCTCTCCAGGCACATCACTGGGGTGCCCGGGATCCAGCGCATTGGGATGGTGCTGGGCGTGGCCATCCAGAAGAGGGCTGTCCCTGGCCTGAAGGCGTTTGAAGAAGCCTATGCCCGGGCAGACAAGGAGGCCCCTAGGCCTTGCACAAGGGCTCCTGGTGCAGCAGCAATCAGCTCTGCAGAGAATGCCAAGCTTTCATGGCACACACGATGCCCAAGCTCAAAGCCTTCTCCATGAGTTCTGCCTACAACGCATACCGGGCTGTGTATGCGGTGGCCCATGGCCTCCACCAGCTCCTGGGCTGTGCCTCTGAGCTCTGTTCCAGGGGCCGAGTCTACCCCTGGCAGCTTTTGGAGCAGATCCACAAGGTGCATTTCCTTCTACACAAGGACACTGTGGCGTTTAATGACAACAGAGATCCCCTCAGTAGCTATAACATAATTGCCTGGGACTGGAATGGACCCAAGTGGACCTTCACGGTCCTCGGTTCCTCCACATGGTCTCCAGTTCAGCTAAACATAAATGAGACCAAAATCCAGTGGCACGGAAAGAACCACCAGGTGCCTAAGTCTGTGTGTTCCAGCGACTGTCTTGAAGGGCACCAGCGAGTGGTTACGGGTTTCCATCACTGCTGCTTTGAGTGTGTGCCCTGTGGGGCTGGGACCTTCCTCAACAAGAGCGAGCTCTACAGATGCCAGCCTTGTGGAACAGAAGAGTGGGCACCTGAGGGAAGCCAGACCTGCTTCCCGCGCACTGTGGTGTTTTTGGCTTTGCGTGAGCACACCTCTTGGGTGCTGCTGGCAGCTAACACGCTGCTGCTGCTGCTGCTGCTTGGGACTGCTGGCCTGTTTGCCTGGCACCTAGACACCCCTGTGGTGAGGTCAGCAGGGGGCCGCCTGTGCTTTCTTATGCTGGGCTCCCTGGCAGCAGGTAGTGGCAGCCTCTATGGCTTCTTTGGGGAACCCACAAGGCCTGCGTGCTTGCTACGCCAGGCCCTCTTTGCCCTTGGTTTCACCATCTTCCTGTCCTGCCTGACAGTTCGCTCATTCCAACTAATCATCATCTTCAAGTTTTCCACCAAGGTACCTACATTCTACCACGCCTGGGTCCAAAACCACGGTGCTGGCCTGTTTGTGATCATCAGCTCAGCGGCCCAGCTGCTTATCTGTCTAACTTGGCTGGTGGTGTGGACCCCACTGCCTGCTAGGGAATACCAGCGCTTCCCCCATCTGGTGATGCTTGAGTGCACAGAGACCAACTCCCTGGGCTTCATACTGGCCTTCCTCTACAATGGCCTCCTCTCCATCAGTGCCTTTGCCTGCAGCTACCTGGGTAAGGACTTGCCAGAGAACTACAACGAGGCCAAATGTGTCACCTTCAGCCTGCTCTTCAACTTCGTGTCCTGGATCGCCTTCTTCACCACGGCCAGCGTCTACGACGGCAAGTACCTGCCTGCGGCCAACATGATGGCTGGGCTGAGCAGCCTGAGCAGCGGCTTCGGTGGGTATTTTCTGCCTAAGTGCTACGTGATCCTCTGCCGCCCAGACCTCAACAGCACAGAGCACTTCCAGGCCTCCATTCAGGACTACACGAGGCGCTGCGGCTCCACCTGA T1R2 SEQUENCES Rat T1R2 aminoacid sequence--SEQ ID NO:7MGPQARTLCLLSLLLHVLPKPGKLVENSDFHLAGDYLLGGLFTLHANVKSISHLSYLQVPKCNEFTMKVLGYNLMQAMRFAVEEINNCSSLLPGVLLGYEMVDVCYLSNNIHPGLYFLAQDDDLLPILKDYSQYMPHVVAVIGPDNSESAITVSNILSHFLIPQITYSAISDKLRDKRHFPSMLRTVPSATHHIEAMVQLMVHFQWNWIVVLVSDDDYGRENSHLLSQRLTKTSDICIAFQEVLPIPESSQVMRSEEQRQLDNILDKLRRTSARVVVVFSPELSLYSFFHEVLRWNFTGFVWIASESWAIDPVLHNLTELRHTGTFLGVTIQRVSIPGFSQFRVRRDKPGYPVPNTTNLRTTCNQDCDACLNTTKSFNNILILSGERVVYSVYSAVYAVAHALHRLLGCNRVRCTKQKVYPWQLLREIWHVNFTLLGNRLFFDQQGDMPMLLDIIQWQWDLSQNPFQSIASYSPTSKRLTYINNVSWYTPNNTVPVSMCSKSCQPGQMKKSVGLHPCCFECLDCMPGTYLNRSADEFNCLSCPGSMWSYKNDITCFQRRPTFLEWHEVPTIVVAILAALGFFSTLAILFIFWRHFQTPMVRSAGGPMCFLMLVPLLLAFGMVPVYVGPPTVFSCFCRQAFFTVCFSICLSCITVRSFQIVCVFKMARRLPSAYSFWMRYHGPYVFVAFITAIKVALVVGNMLATTINPIGRTDPDDPNIMILSCHPNYRNGLLFNTSMDLLLSVLGFSFAYMGKELPTNYNEAKFITLSMTFSFTSSISLCTFMSVHDGVLVTIMDLLVTVLNFLAIGLGYFGPKCYMILFYPERNTSAYFNSMIQGYTMRKS Mouse T1R2 amino acidsequence--SEQ ID NO:8MGPQARTLHLLFLLLHALPKPVMLVGNSDFHLAGDYLLGGLFTLHANVKSVSHLSYLQVPKCNEYNMKVLGYNLMQANRFAVEETNNCSSLLPGVLLGYEMVDVCYLSNNIQPGLYFLSQIDDELPILKDYSQYRPQVVAVIGPDNSESAITVSNILSYFLVPQVTYSAITDKLQDKRRFPAMLRTVPSATHHIEAMVQLMVHFQWNWIVVLVSDDDYGRENSHLLSQRLTNTGDICIAFQEVLPVPEPNQAVRPEEQDQLDNILDKLRRTSARVVVIFSPELSLHNFFREVLRWNFTGFVWIASESWAIDPVLHNLTELRHTGTELGVTIQRVSIPGFSQFRVRHDKPGYRMPNETSLRTTCNQDCDACMNITESFNNVLMLSGERVVYSVYSAVYAVAHTLHRLLHCNQVRCTKQIVYPWQLLREIWHVNFTLLGNQLFFDEQGDMPMLLDIIQWQWGLSQNPFQSIASYSPTETRLTYISNVSWYTPNNTVPISMCSKSCQPGQMKKPIGLHPCCFECVDCPPDTYLNRSVDEFNCLSCPGSMWSYKNNIACFKRRLAFLEWHEVPTIVVTILAALGFISTLAILLIFWRHFQTPMVRSAGGPMCFLMLVPLLLAFGMVPVYVGPPTVFSCFCRQAFFTVCFSVCLSCITVRSFQIVCVFKMARRLPSAYGFWMRYHGPYVFVAEITAVKVALVAGNMLATTINPIGRTDPDDPNIIILSCHPNYRNGLLFNTSMDLLLSVLGFSFAYVGKELPTNYNEAKFITLSMTFSFTSSISLCTFMSVHDGVLVTIMDLLVTVLNFLAIGLGYFGPKCYMILFYPERNTSAYFNSMIQGYTMRKS Human T1R2 amino acidsequence--SEQ ID NO:9MGPRAKTICSLFFLLWVLAEPAENSDFYLPGDYLLGGLFSLHANMKGIVHLNFLQVPMCKEYEVKVIGYNLMQAMRFAVEEINNDSSLLPGVLLGYEIVDVCYISNNVQPVLYFLAHEDNLLPIQEDYSNYISRVVAVIGPDNSESVMTVANFLSLFLLPQITYSAISDELRDKVRFPALLRTTPSADHHVEAMVQLMLHFRWNWIIVLVSSDTYGRDNGQLLGERVARRDICIAFQETLPTLQPNQNMTSEERQRLVTIVDKLQQSTARVVVVFSPDLTLYHFFNEVLRQNFTGAVWIASESWAIDPVLHNLTELGHLGTFLGITIQSVPIPGFSEFREWGPQAGPPPLSRTSQSYTCNQECDNCLNATLSFNTILRLSGERVVYSVYSAVYAVAHALHSLLGCDKSTCTKRVVYPWQLLEEIWKVNFTLLDHQIFFDPQGDVALHLEIVQWQWDRSQNPFQSVASYYPLQRQLKNIQDISWHTVNNTIPMSMCSKRCQSGQKKKPVGIHVCCFECIDCLPGTFLNHTEDEYECQACPNNEWSYQSETSCFKRQLVFLEWHEAPTIAVALLAALGFLSTLAILVIFWRHFQTPIVRSAGGPMCELMLTLLLVAYMVVPVYVGPPKVSTCLCRQALFPLCFTICISCIAVRSFQIVCAFKMASRFPRAYSYWVRYQGPYVSMAFITVLKMVIVVIGMLARPQSHPRTDPDDPKITIVSCNPNYRNSLLFNTSLDLLLSVVGFSFAYMGKELPTNYNEAKFITLSMTFYFTSSVSLCTFMSAYSGVLVTIVDLLVTVLNLLAISLGYFGPKCYMILFYPERNTPAYFNSMIQGYTMRRD Rat T1R2 nucleotide sequence--SEQ IDNO:10 CACTTTGCTGTCATGGGTCCCCAGGCAAGGACACTCTGCTTGCTGTCTCTCCTGCTGCATGTTCTGCCTAAGCCAGGCAAGCTGGTAGAGAACTCTGACTTCCACCTGGCCGGGGACTACCTCCTGGGTGGCCTCTTTACCCTCCATGCCAACGTGAAGAGCATCTCCCACCTCAGCTACCTGCAGGTGCCCAAGTGCAATGAGTTCACCATGAAGGTGTTGGGCTACAACCTCATGCAGGCCATGCGTTTCGCTGTGGAGGAGATCAACAACTGTAGCTCCCTGCTACCCGGCGTGCTGCTCGGCTACGAGATGGTGGATGTCTGTTACCTCTCCAACAATATCCACCCTGGGCTCTACTTCCTGGCACAGGACGACGACCTCCTGCCCATCCTCAAAGACTACAGCCAGTACATGCCCCACGTGGTGGCTGTCATTGGCCCCGACAACTCTGAGTCCGCCATTACCGTGTCCAACATTCTCTCTCATTTCCTCATCCCACAGATCACATACAGCGCCATCTCCGACAAGCTGCGGGACAAGCGGCACTTCCCTAGCATGCTACGCACAGTGCCCAGCGCCACCCACCACATCGAGGCCATGGTGCAGCTGATGGTTCACTTCCAATGGAACTGGATTGTGGTGCTGGTGAGCGACGACGATTACGGCCGCGAGAACAGCCACCTGTTGAGCCAGCGTCTGACCAAAACGAGCGACATCTGCATTGCCTTCCAGGAGGTTCTGCCCATACCTGAGTCCAGCCAGGTCATGAGGTCCGAGGAGCAGAGACAACTGGACAACATCCTGGACAAGCTGCGGCGGACCTCGGCGCGCGTCGTGGTGGTGTTCTCGCCCGAGCTGAGCCTGTATAGCTTCTTTCACGAGGTGCTCCGCTGGAACTTCACGGGTTTTGTGTGGATCGCCTCTGAGTCCTGGGCTATCGACCCAGTTCTGCATAACCTCACGGAGCTGCGCCACACGGGTACTTTTCTGGGCGTCACCATCCAGAGGGTGTCCATCCCTGGCTTCAGTCAGTTCCGAGTGCGCCGTGACAAGCCAGGGTATCCCGTGCCTAACACGAGCAAGCTGCGGACGACCTGCAACCAGGACTGTGACGCCTGCTTGAACACCACCAAGTCCTTCAACAACATCCTTATACTTTCGGGGGAGCGCGTGGTCTACAGCGTGTACTCGGCAGTTTACGCGGTGGCCCATGCCCTCCACAGACTCCTCGGCTGTAACCGGGTCCGCTGCACCAAGCAAAAGGTCTACCCGTGGCAGCTACTCAGGGAGATCTGGCACGTCAACTTCACGCTCCTGGGTAACCGGCTCTTCTTTGACCAACAAGGGGACATGCCGATGCTCTTGGACATCATCCAGTGGCAGTGGGACCTGAGCCAGAATCCCTTCCAAAGCATCGCCTCCTATTCTCCCACCAGCAAGAGGCTAACCTACATTAACAATGTGTCCTGGTACACCCCCAACAACACGGTCCCTGTCTCCATGTGTTCCAAGAGCTGCCAGCCAGGGCAAATGAAAAAGTCTGTGGGCCTCCACCCTTGTTGCTTCGAGTGCTTGGATTGTATGCCAGGCACCTACCTCAACCGCTCAGCAGATGAGTTTAACTGTCTGTCCTGCCCGGGTTCCATGTGGTCCTACAAGAACGACATCACTTGCTTCCAGCGGCGGCCTACCTTCCTGGAGTGGCACGAAGTGCCCACCATCGTGGTGGCCATACTGGCTGCCCTGGGCTTCTTCAGTACACTGGCCATTCTTTTCATCTTCTGGAGACATTTCCAGACACCCATGGTGCGCTCGGCCGGTGGCCCCATGTGCTTCCTGATGCTCGTGCCCCTGCTGCTGGCGTTTGGGATGGTGCCCGTGTATGTGGGGCCCCCCACGGTCTTCTCATGCTTCTGCCGACAGGCTTTCTTCACCGTCTGCTTCTCCATCTGCCTATCCTGCATCACCGTGCGCTCCTTCCAGATCGTGTGTGTCTTCAAGATGGCCAGACGCCTGCCAAGTGCCTACAGTTTTTGGATGCGTTACCACGGGCCCTATGTCTTCGTGGCCTTCATCACGGCCATCAAGGTGGCCCTGGTGGTGGGCAACATGCTGGCCACCACCATCAACCCCATTGGCCGGACCGACCCGGATGACCCCAACATCATGATCCTCTCGTGCCACCCTAACTACCGCAACGGGCTACTGTTCAACACCAGCATGGACTTGCTGCTGTCTGTGCTGGGTTTCAGCTTCGCTTACATGGGCAAGGAGCTGCCCACCAACTACAACGAAGCCAAGTTCATCACTCTCAGCATGACCTTCTCCTTCACCTCCTCCATCTCCCTCTGCACCTTCATGTCTGTGCACGACGGCGTGCTGGTCACCATCATGGACCTCCTGGTCACTGTGCTCAACTTCCTGGCCATCGGCTTGGGATACTTTGGCCCCAAGTGTTACATGATCCTTTTCTACCCGGAGCGCAACACCTCAGCCTATTTCAATAGCATGATCCAGGGCTACACCATGAGGAAGAGCTAGCTCCGCCCACCGGCCTCAGCAGCAGAGCCCCCGGCCACGTTAATGGTGTTCCTCTGCCATTCTCTGCAGCGTAGCTATTTTTACCCACATAGCGCTTAAAATACCCATGATGCACTCTCCCCCGACCCCCAAGCCATTTCACTGGCCAGGACCTACCACCCACTTATAGATGAAACCACCAAGGCGCCCTATGGGGCTCCAAGGATGGCCTACCACTGCCATCTGGTGGTCACAGTGAGCACATGCGGGCCGTGGCCCATGGCTCCCAGCCAGCTGGTGGCTAGTGGCTGTGAGGCCAGATGTCTGTGTATCTGAGTTCCTGGGAAGCAGAGACTGGGGCTCCTGTGTTCTAATGGTCAGATGGGCATCATGGGCCCTTCATTATTGCTTACGAATAAACTTCCCTCCGGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Mouse T1R2 nucleotide sequence--SEQ ID NO:11ATGGGACCCCAGGCGAGGACACTCCATTTGCTGTTTCTCCTGCTGCATGCTCTGCCTAAGCCAGTCATGCTGGTAGGGAACTCCGACTTTCACCTGGCTGGGGACTACCTCCTGGGTGGCCTCTTTACCCTCCATGCCAACGTGAAGAGTGTCTCTCACCTCAGCTACCTGCAGGTGCCCAAGTGCAATGAGTACAACATGAAGGTGTTGGGCTACAACCTCATGCAGGCCATGCGATTCGCCGTGGAGGAAATCAACAACTGTAGCTCTTTGCTGCCCGGCGTGCTCCTCGGCTACGAGATGGTGGATGTCTGCTACCTCTCCAACAATATCCAGCCTGGGCTCTACTTCCTGTCACAGATAGATGACTTCCTGCCCATCCTCAAAGACTACAGCCAGTACAGGCCCCAAGTGGTGGCTGTTATTGGCCCAGACAACTCTGAGTCTGCCATCACCGTGTCCAACATTCTCTCCTACTTCCTCGTGCCACAGGTCACATATAGCGCCATCACCGACAAGCTGCAAGACAAGCGGCGCTTCCCTGCCATGCTGCGCACTGTGCCCAGCGCCACCCACCACATCGAGGCCATGGTGCAACTGATGGTTCACTTCCAGTGGAACTGGATCGTGGTGCTGGTGAGCGATGACGATTATGGCCGAGAGAACAGCCACCTGCTGAGCCAGCGTCTGACCAACACTGGCGACATCTGCATTGCCTTCCAGGAGGTTCTGCCCGTACCAGAACCCAACCAGGCTGTGAGGCCTGAGGAGCAGGACCAACTGGACAACATCCTGGACAAGCTGCGGCGGACTTCGGCGCGTGTGGTGGTGATATTCTCGCCGGAGCTGAGCCTGCACAACTTCTTCCGTGAGGTGCTGCGCTGGAACTTCACGGGCTTTGTGTGGATTGCCTCTGAGTCCTGGGCCATCGACCCTGTTCTACACAACCTCACAGAGCTGCGCCACACGGGCACTTTCCTGGGTGTCACCATCCAGAGGGTGTCCATCCCTGGCTTCAGCCAGTTCCGAGTGCGCCATGACAAGCCAGGGTATCGCATGCCTAACGAGACCAGCCTGCGGACTACCTGTAACCAGGACTGCGACGCCTGCATGAACATCACTGAGTCCTTCAACAACGTTCTCATGCTTTCGGGGGAGCGTGTGGTCTACAGCGTGTACTCGGCCGTCTACGCGGTGGCCCACACCCTCCACAGACTCCTCCACTGCAATCAGGTCCGCTGCACCAAGCAAATCGTCTATCCATGGCAGCTACTCAGGGAGATCTGGCATGTCAACTTCACGCTCCTGGGCAACCAGCTCTTCTTCGACGAACAAGGGGACATGCCGATGCTCCTGGACATCATCCAGTGGCAGTGGGGCCTGAGCCAGAACCCCTTCCAAAGCATCGCCTCCTACTCCCCCACCGAGACGAGGCTGACCTACATTAGCAATGTGTCCTGGTACACCCCCAACAACACGGTCCCCATATCCATGTGTTCTAAGAGTTGCCAGCCTGGGCAAATGAAAAAACCCATAGGCCTCCACCCATGCTGCTTCGAGTGTGTGGACTGTCCGCCGGACACCTACCTCAACCGATCAGTAGATGAGTTTAACTGTCTGTCCTGCCCGGGTTCCATGTGGTCTTACAAGAACAACATCGCTTGCTTCAAGCGGCGGCTGGCCTTCCTGGAGTGGCACGAAGTGCCCACTATCGTGGTGACCATCCTGGCCGCCCTGGGCTTCATCAGTACGCTGGCCATTCTGCTCATCTTCTGGAGACATTTCCAGACGCCCATGGTGCGCTCGGCGGGCGGCCCCATGTGCTTCCTGATGCTGGTGCCCCTGCTGCTGGCGTTCGGGATGGTCCCCGTGTATGTGGGCCCCCCCACGGTCTTCTCCTGTTTCTGCCGCCAGGCTTTCTTCACCGTTTGCTTCTCCGTCTGCCTCTCCTGCATCACGGTGCGCTCCTTCCAGATTGTGTGCGTCTTCAAGATGGCCAGACGCCTGCCAAGCGCCTACGGTTTCTGGATGCGTTACCACGGGCCCTACGTCTTCGTGGCCTTCATCACGGCCGTCAAGGTGGCCCTGGTGGCGGGCAACATGCTGGCCACCACCATCAACCCCATTGGCCGGACCGACCCCGATGACCCCAATATCATAATCCTCTCCTGCCACCCTAACTACCGCAACGGGCTACTCTTCAACACCAGCATGGACTTGCTGCTGTCCGTGCTGGGTTTCAGCTTCGCGTACGTGGGCAAGGAACTGCCCACCAACTACAACGAAGCCAAGTTCATCACCCTCAGCATGACCTTCTCCTTCACCTCCTCCATCTCCCTCTGCACGTTCATGTCTGTCCACGATGGCGTGCTGGTCACCATCATGGATCTCCTGGTCACTGTGCTCAACTTTCTGGCCATCGGCTTGGGGTACTTTGGCCCCAAATGTTACATGATCCTTTTCTACCCGGAGCGCAACACTTCAGCTTATTTCAATAGCATGATTCAGGGCTACACGATGAGGAAGAGCTAG Human T1R2nucleotide sequence--SEQ ID NO:12ATCACCTACAGCGCCATCAGCGATGAGCTGCGAGACAAGGTGCGCTTCCCGGCTTTGCTGCGTACCACACCCAGCGCCGACCACCACGTCGAGGCCATGGTGCAGCTGATGCTCCACTTCCGCTGGAACTGGATCATTGTGCTGGTGAGCAGCGACACCTATGGCCGCGACAATGGCCAGCTGCTTGGCGAGCGCGTGGCCCGGCGCGACATCTGCATCGCCTTCCAGGAGACGCTGCCCACACTGCAGCCCAACCAGAACATGACGTCAGAGGAGCGCCAGCGCCTGGTGACCATTGTGGACAAGCTGCAGCAGAGCACAGCGCGCGTCGTGGTCGTGTTCTCGCCCGACCTGACCCTGTACCACTTCTTCAATGAGGTGCTGCGCCAGAACTTCACGGGCGCCGTGTGGATCGCCTCCGAGTCCTGGGCCATCGACCCGGTCCTGCACAACCTCACGGAGCTGGGCCACTTGGGCACCTTCCTGGGCATCACCATCCAGAGCGTGCCCATCCCGGGCTTCAGTGAGTTCCGCGAGTGGGGCCCACAGGCTGGGCCGCCACCCCTCAGCAGGACCAGCCAGAGCTATACCTGCAACCAGGAGTGCGACAACTGCCTGAACGCCACCTTGTCCTTCAACACCATTCTCAGGCTCTCTGGGGAGCGTGTCGTCTACAGCGTGTACTCTGCGGTCTATGCTGTGGCCCATGCCCTGCACAGCCTCCTCGGCTGTGACAAAAGCACCTGCACCAAGAGGGTGGTCTACCCCTGGCAGCTGCTTGAGGAGATCTGGAAGGTCAACTTCACTCTCCTGGACCACCAAATCTTCTTCGACCCGCAAGGGGACGTGGCTCTGCACTTGGAGATTGTCCAGTGGCAATGGGACCGGAGCCAGAATCCCTTCCAGAGCGTCGCCTCCTACTACCCCCTGCAGCGACAGCTGAAGAACATCAAGACATCTCTGCACACCGTCAACAACACGATCCCTATGTCCATGTGTTCCAAGAGGTGCCAGTCAGGGCAAAAGAAGAAGCCTGTGGGCATCCACGTCTGCTGCTTCGAGTGCATCGACTGCCTTCCCGGCACCTTCCTCAACCACACTGAATGCCCGAATAACGAGTGGTCCTACCAGAGTGAGACCTCCTGCTTCAAGCGGCAGCTGGTCTTCCTGGAATGGCATGAGGCACCCACCATCGCTGTGGCCCTGCTGGCCGCCCTGGGCTTCCTCAGCACCCTGGCCATCCTGGTGATATTCTGGAGGCACTTCCAGACACCCATAGTTCGCTCGGCTGGGGGCCCCATGTGCTTCCTGATGCTGACACTGCTGCTGGTGGCATACATGGTGGTCCCGGTGTACGTGGGGCCGCCCAAGGTCTCCACCTGCCTCTGCCGCCAGGCCCTCTTTCCCCTCTGCTTCACAATTTGCATCTCCTGTATCGCCGTGCGTTCTTTCCAGATCGTCTGCGCCTTCAAGATGGCCAGCCGCTTCCCACGCGCCTACAGCTACTGGGTCCGCTACCAGGGGCCCTACGTCTCTATGGCATTTATCACGGTACTCAAAATGGTCATTGTGGTAATTGGCATGCTGGCACGGCCTCAGTCCCACCCCCGTACTGACCCCGATGACCCCAAGATCACAATTGTCTCCTGTAACCCCAACTACCGCAACAGCCTGCTGTTCAACACCAGCCTGGACCTGCTGCTCTCAGTGGTGGGTTTCAGCTTCGCCTACATGGGCAAAGAGCTGCCCACCAACTACAACGAGGCCAAGTTCATCACCCTCAGCATGACCTTCTATTTCACCTCATCCGTCTCCCTCTGCACCTTCATGTCTGCCTACAGCGGGGTGCTGGTCACCATCGTGGACCTCTTGGTCACTGTGCTCAACCTCCTGGCCATCAGCCTGGGCTACTTCGGCCCCAAGTGCTACATGATCCTCTTCTACCCGGAGCGCAACACGCCCGCCTACTTCAACAGCATGATCCAGGGCTACACCATGAGGAGGGACTAG TIR3 SEQUENCES Human T1R3 genomic nucleotidesequence--SEQ ID NO:13GCTCACTCCATGTGAGGCCCCAGTCGGGGCAGCCACCTGCCGTGCCTGTTGGAAGTTGCCTCTGCCATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTACAGAGGTGGGACGGCCTGGGTCGGGGTCAGGGTGACCAGGTCTGGGGTGCTCCTGAGCTGGGGCCGAGGTGGCCATCTGCGGTTCTGTGTGGCCCCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGGTGGCGCCCCCCACCATCACCCACCCCCACCCAGCCCTGCCCGTGGGAGCCCCTGTGTCAGGAGATGCCTCTTGGCCCTTGCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGGTGAGCCCGGGAGATGGGGGTGTGCTGTCCTCTGCATGTGCCCAGGCCACCAGGCACGGCCACCACGCCTGAGCTGGAGGTGGCTGGCGGCTCAGCCCCGTCCCCCGCCCGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGGTGAGGTGAGGGTGGGTGTGCCAGGCGTGCCCGTGGTAGCCCCCGCGGCAGGGCGCAGCCTGGGGGTGGGGGCCGTTCCAGTCTCCCGTGGGCATGCCCAGCCGAGCAGAGCCAGACCCCAGGCCTGTGCGCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGGTGAGCCGCCTTCCCGGCAGGCGGGGGTGGGAACGCAGCAGGGGAGGGTCCTGCCAAGTCCTGACTCTGAGACCAGAGCCCACAGGGTACAAGACGAACACCCAGCGCCCTTCTCCTCTCTCACAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGACCCAACCCTGTGATCT Human T1R3 cds nucleotidesequence--SEQ ID NO:14atgctgggccctgctgtcctgggcctcagcctctgggctctcctgcaccctgggacgggggccccattgtgcctgtcacagcaacttaggatgaagggggactacgtgctgggggggctgttccccctgggcgaggccgaggaggctggcctccgcagccggacacggcccagcagccctgtgtgcaccaggttctcctcaaacggcctgctctgggcactggccatgaaaatggccgtggaggagatcaacaacaagtcggatctgctgcccgggctgcgcctgggctacgacctctttgatacgtgctcggagcctgtggtggccatgaagcccagcctcatgttcctggccaaggcaggcagccgcgacatcgccgcctactgcaactacacgcagtaccagccccgtgtgctggctgtcatcgggccccactcgtcagagctcgccatggtcaccggcaagttcttcagcttcttcctcatgccccagGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTCGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGGTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGA Human T1R3 amino acid sequence--SEQ ID NO:15MLGPAVLGLSLWALLHPGTGAPLCLSQQLRMKGDYVLGGLFPLGEAEEAGLRSRTRPSSPVCTRFSSNGLLWALAMKMAVEEINNKSDLLPGLRLGYDLFDTCSEPVVAMKPSLMFLAKAGSRDIAAYCNYTQYQPRVLAVIGPHSSELAMVTGKFFSFFLMPQVSYGASMELLSARETFPSFFRTVPSDRVQLTAAAELLQEFGWNWVAALGSDDEYGRQGLSIFSALAAARGICIAHEGLVPLPRADDSRLGKVQDVLHQVNQSSVQVVLLFASVHAAHALFNYSISSRLSPKVWVASEAWLTSDLVMGLPGMAQMGTVLGFLQRGAQLHEFPQYVKTHLALATDPAFCSALGEREQGLEEDVVGQRCPQCDCITLQNVSAGLNHHQTFSVYAAVYSVAQALHNTLQCNASGCPAQDPVKPWQLLENMYNLTFHVGGLPLRFDSSGNVDMEYDLKLWVWQGSVPRLHDVGRFNGSLRTERLKIRWHTSDNQKPVSRCSRQCQEGQVRRVKGFHSCCYDCVDCEAGSYRQNPDDIACTFCGQDEWSPERSTRCFRRRSRFLAWGEPAVLLLLLLLSLALGLVLAALGLFVHHRDSPLVQASGGPLACFGLVCLGLVCLSVLLFPGQPSPARCLAQQPLSHLPLTGCLSTLFLQAAEIFVESELPLSWADRLSGCLRGPWAWLVVLLAMLVEVALCTWYLVAFPPEVVTDWHMLPTEALVHCRTRSWVSFGLAHATNATLAFLCFLGTFLVRSQPGCYNRARGLTFAMLAYFITWVSFVPLLANVQVVLRPAVQMGALLLCVLGILAAFHLPRCYLLMRQPGLNTPEFFLGGGPGDAQGQNDGNTGNQGKHE Mouse T1R3 Sac non taster129 genomic nucleotide sequence-- SEQ ID NO:16ACATCTGTGGCTCCAACCCCACACACCCATCTATTGTTAGTGCTGGAGACTTCTACCTACCATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCGACCGAGGAGGCCACTCTCAACCAGAGAGCACAACCCAACAGCACCCTGTGTA ACAGGTATGGAGGCTAGTAGCTGGGGTGGGAGTGAACCGAAGCTTGGCAGCTTTGGCTCCGTGGTACTACCAATCTGGGGAAGGGGTGGTGATCAGTTTCCATGTGGCCTCAG GTTCTCACCCCTCGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTATGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGTCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAG GTGAGCCCACTTCCTTTGTGTTCTCAACCGATTGCACCCATTGAGCTCTCACATCAGAAAGTGCTTCTTGATCACCACAG GTCAGCTATAGCGCCAGCATGGATCGGCTAAGTGACCGGGAAACGTTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGAACTGGGTGGCCGCCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTAGTCTGGCCAATGCACGAGGTATCTGCATCGCACATGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTGGATGTGCTACGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCATGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGGTTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACAGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAACCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGTCTCACATTGCCACGTATCAGAACATGTTCTACCCTGGCAG GTAAGGGTAGGGTTTTTTGCTGGGTTTTGCCTGCTCCTGCAGGAACACTGAACCAGGCAGAGCCAAATCATGTTGTGACTGGAGAGGCCTTACCCTGACTCCACTCCACAG CTCCTGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTTTGATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAGTCTAAAATGTACTGGCCAGGCAACCAG GTAAGGACAAGACAGGCAAAAAGGATGGTGGGTAGAAGCTTGTCGGTCTTGGGCCAGTGCTAGCCAAGGGGAGGCCTAACCCAAGGCTCCATGTCCAG GTGCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCATCCAG GTGAACCGTCTTCCCTAGACAGTCTGCACAGCCGGGCTAGGGGGCAGAAGCATTCAAGTCTGGCAAGCGCCCTCCCGCGGGGCTAATGTGGAGACAGTTACTGTGGGGGCTGGCTGGGGAGGTCGGTCTCCCATCAGCAGACCCCACATTACTTTTCTTCCTTCCATCACTACAG ATGACTTCACCTGTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGCGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGACCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTGCTATGTGCTTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGAACACAATGAATGACCACTGACCCGTGACCTTCCCTTTAGGGA Mouse T1R3 Sac non taster 129 cds nucleotide sequence--SEQ ID NO:17ATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCGACCGAGGAGGCCACTCTCAACCAGAGAGCACAACCCAACAGCACCCTGTGTAACAGGTTCTCACCCCTCGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTATGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGTCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGCGCCAGCATGGATCGGCTAAGTGACCGGGAAACGTTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGAACTGGGTGGCCGCCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTAGTCTGGCCAATGCACGAGGTATCTGCATCGCACATGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTGGATGTGCTACGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCATGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGGTTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACAGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAACCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGTCTCACATTGCCACGTATCAGAACATGTTCTACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTTTGATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAGTCTAAAATGTACTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGCGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGACCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTGCTATGTGCTTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGAACACAATGAATGA Mouse T1R3 Sac non taster 129 aminoacid sequence-- SEQ ID NO:18MPALAIMGLSLAAFLELGMGASLCLSQQFKAQGDYILGGLFPLGSTEEATLNQRAQPNSTLCNRFSPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKSSLMFLAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELALITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGLSIFSSLANARGICIAHEGLVPQHDTSGQQLGKVLDVLRQVNQSKVQVVVLFASARAVYSLFSYSIHHGLSPKVWVASESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETHLALAADPAFCASLNAELDLEEHVMGQRCPQCDDIMLQNLSSGLLQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHVSEHVLPWQLLENMYNMSFHARDLTLQFDAEGNVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQQSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTCTPCNQDQWSPEKSTACLPRRPKFLAWGEPVVLSLLLLLCLVLGLALAALGLSVHHWDSPLVQASGGSQFCFGLICLGLFCLSVLLFPGRPSSASCLAQQPMAHLPLTGCLSTLFLQAAETFVESELPLSWANWLCSYLRGLWAWLVVLLATFVEAALCAWYLTAFPPEVVTDWSVLPTEVLEHCHVRSWVSLGLVHITNAMLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFITWVSFVPLLANVQVAYQPAVQMGAILVCALGILVTFHLPKCYVLLWLPKLNTQEFFLGRNAKKAADENSGGGEAAQEHNE Mouse T1R3 Sactaster SWR cds nucleotide sequence-- SEQ ID NO:19ATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCAACCGAGGAGGCCACTCTCAACCAGAGAACACAACCCAACAGCATCCTGTGTAACAGGTTCTCACCCCTCGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTATGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGTCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGCGCCAGCATGGATCGGCTAAGTGACCGGGAAACGTTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGAACTGGGTGGCCGCCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTAGTCTGGCCAATGCACGAGGTATCTGCATCGCACATGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTGGATGTGCTATGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCATGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGGTTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACAGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAACCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGTCTCACATTGCCATGTATCAGAACATGTTCTACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTTTGATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAGTCTAAAATGTACTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGCGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTCGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGACCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTGCTATGTGCTTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGAACACAATGAATGA Mouse T1R3 Sac taster SWR amino acidsequence--SEQ ID NO:20MPALAIMGLSLAAFLELGMGASLCLSQQFKAQGDYILGGLFPLGSTEEATLNQRTQPNSILCNRFSPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKSSLMFLAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELALITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGLSIFSSLANARGICIAHEGLVPQHDTSGQQLGKVLDVLCQVNQSKVQVVVLFASARAVYSLFSYSIHHGLSPKVWVASESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETHLALAADPAFCASLNAELDLEEHVMGQRCPQCDDIMLQNLSSGLLQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHVSEHVLPWQLLENMYNMSFHARDLTLQFDAEGNVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQQSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTCTPCNQDQWSPEKSTACLPRRPKFLAWGEPVVLSLLLLLCLVLGLALAALGLSVHHWDSPLVQASGGSQFCFGLICLGLFCLSVLLFPGRPSSASCLAQQPMAHLPLTGCLSTLFLQAAETFVESELPLSWANWLCSYLRGLWAWLVVLSATFVEAALCAWYLTAFPPEVVTDWSVLPTEVLEHCHVRSWVSLGLVHITNAMLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFITWVSFVPLLANVQVAYQPAVQMGAILVCALGILVTFHLPKCYVLLWLPKLNTQEFFLGRNAKKAADENSGGGEAAQEHNE Mouse T1R3 Sactaster c57 genomic nucleotide sequence-- SEQ ID NO:21CCCACACACCCACCCATTGTTAGTGCTGGAGACTTCTACCTACCATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCAACCGAGGAGGCCACTCTCAACCAGAGAACACAACCCAACAGCATCCCGTGCAACAGGTATGGAGGCTAGTAGCTGGGGTGGGAGTGAACCGAAGCTTGGCAGCTTTGGCTCCGTGGTACTACCAATCTGGGAAGAGGTGGTGATCAGTTTCCATGTGGCCTCAGGTTCTCACCCCTTGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTATGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGTCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTGAGCCCACTTCCTTTGTGTTCTCAACCGATTGCACCCATTGAGCTCTCATATCAGAAAGTGCTTCTTGATCACCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGGGAAACGTTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGAACTGGGTGGCCGCCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTAGTCTGGCCAATGCACGAGGTATCTGCATCGCACATGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTGGATGTACTACGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCATGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGGTTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACGGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAACCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGTCTCACATTGCCACGTATCAGAACATGTTCTACCCTGGCAGGTAAGGGTAGGGTTTTTTGCTGGGTTTTGCCTGCTCCTGCAGGAACACTGAACCAGGCAGAGCCAAATCTTGTTGTGACTGGAGAGGCCTTACCCTGACTCCACTCCACAGCTCCTGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTTTGATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAGTCTAAAATGTACTGGCCAGGCAACCAGGTAAGGACAAGACAGGCAAAAAGGATGGTGGGTAGAAGCTTGTCGGTCTTGGGCCAGTGCTAGCCAAGGGGAGGCCTAACCCAAGGCTCCATGTACAGGTGCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCATCCAGGTGAACCGTCTTCCCTAGACAGTCTGCACAGCCGGGCTAGGGGGCAGAAGCATTCAAGTCTGGCAAGCGCCCTCCCGCGGGGCTAATGTGGAGACAGTTACTGTGGGGGCTGGCTGGGGAGGTCGGTCTCCCATCAGCAGACCCCACATTACTTTTCTTCCTTCCATCACTACAGATGACTTCACCTGTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGTGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGGCGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGATCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTGCTATGTGCTTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGGACACAATGAATGACCACTGA Mouse T1R3 Sac taster C57 cdsnucleotide sequence-- SEQ ID NO:22ATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCAACCGAGGAGGCCACTCTCAACCAGAGAACACAACCCAACAGCATCCCGTGCAACAGGTTCTCACCCCTTGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTATGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGTCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGGGAAACGTTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGAACTGGGTGGCCGCCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTAGTCTGGCCAATGCACGAGGTATCTGCATCGCACATGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTGGATGTACTACGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCATGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGGTTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACGGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAACCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGTCTCACATTGCCACGTATCAGAACATGTTCTACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTTTCATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAGTCTAAAATGTACTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGTGGCTCACAGTTCTGCTTTGGCCTGATCTCCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGGCGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGATCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTGCTATGTGCTTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGGACACAATGAATGA Mouse T1R3 Sac taster C57 amino acidsequence--SEQ ID NO:23MPALAIMGLSLAAFLELGMGASLCLSQQFKAQGDYILGGLFPLGSTEEATLNQRTQPNSIPCNRFSPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKSSLMFLAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELALITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGLSIFSSLANARGICIAHEGLVPQHDTSGQQLGKVLDVLRQVNQSKVQVVVLFASARAVYSLFSYSIHHGLSPKVWVASESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETHLALAADPAFCASLNAELDLEEHVMGQRCPRCDDIMLQNLSSGLLQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHVSEHVLPWQLLENMYNMSFHARDLTLQFDAEGNVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQQSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTCTPCNQDQWSPEKSTACLPRRPKFLAWGEPVVLSLLLLLCLVLGLALAALGLSVHHWDSPLVQASGGSQFCFGLICLGLFCLSVLLFPGRPSSASCLAQQPMAHLPLTGCLSTLFLQAAETFVESELPLSWANWLCSYLRGLWAWLVVLLATFVEAALCAWYLIAFPPEVVTDWSVLPTEVLEHCHVRSWVSLGLVHITNAMLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFITWVSFVPLLANVQVAYQPAVQMGAILVCALGILVTFHLPKCYVLLWLPKLNTQEFFLGRNAKKAADENSGGGEAAQGHNE Rat T1R3 CDSnucleotide sequence--SEQ ID NO:24ATGCCGGGTTTGGCTATCTTGGGCCTCAGTCTGGCTGCTTTCCTGGAGCTTGGGATGGGGTCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTATATATTGGGTGGACTATTTCCCCTGGGCACAACTGAGGAGGCCACTCTCAACCAGAGAACACAGCCCAACGGCATCCTATGTACCAGGTTCTCGCCCCTTGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTAGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGACTGGGCTATGACCTGTTTGACACATGCTCAGAGCCAGTGGTCACCATGAAGCCCAGCCTCATGTTCATGGCCAAGGTGGGAAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATTGGTCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGGGAAACATTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCCGTTGTGACACTGTTGCAGAATTTCAGCTGGAACTGGGTGGCTGCCTTAGGTAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTGGTCTGGCCAACTCACGAGGTATCTGCATTGCACACGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAATTGGGCAAGGTGGTGGATGTGCTACGCCAAGTGAACCAAAGCAAAGTACAGGTGGTGGTGCTGTTTGCATCTGCCCGTGCTGTCTACTCCCTTTTTAGCTACAGCATCCTTCATGACCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCCTGGCTGACCTCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTTCTTGGGTTTCTGCAGCGCGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCGCCTTGCCCTAGCTGCTGACCCAACATTCTGTGCCTCCCTGAAAGCTGAGTTGGATCTGGAGGAGCGCGTGATGGGGCCACGCTGTTCACAATGTGACTACATCATGCTACAGAACCTGTCATCTGGGCTGATGCAGAACCTATCAGCTGGGCAGTTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAGGCCCTTCACAACACCCTGCAGTGCAATGTCTCACATTGCCACACATCAGAGCCTGTTCAACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCGTGCTCGAGACTTGACACTGCAGTTTGATGCCAAAGGGAGTGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTACTACATACTGTAGGCACCTTCAACGGCACCCTTCAGCTGCAGCACTCGAAAATGTATTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGCTCCCGGCAGTGCAAAGATGGCCAGGTGCGCAGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGTGTGGACTGCAAGGCAGGGAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTGGCAAGGATCAGTGGTCCCCAGAAAAAAGCACAACCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGCTGTGCTGTCACTTCTCCTGCTGCTTTGCCTGGTGCTGGGCCTGACACTGGCTGCCCTGGGGCTCTTTGTCCACTACTGGGACAGCCCTCTTGTTCAGGCCTCAGGTGGGTCACTGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGACCACGCTCTGCCAGCTGCCTTGCCCAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCCGAGATCTTTGTGGAGTCTGAGCTGCCACTGAGTTGGGCAAACTGGCTCTGCAGCTACCTTCGGGGCCCCTGGGCTTGGCTGGTGGTACTGCTGGCCACTCTTGTGGAGGCTGCACTATGTGCCTGGTACTTGATGGCTTTCCCTCCAGAGGTGGTGACAGATTGGCAGGTGCTGCCCACGGAGGTACTGGAACACTGCCGCATGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAGTGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGTCGCTATAACCGTGCCCGTGGCCTCACCTTCGCCATGCTAGCTTATTTCATCATCTGGGTCTCTTTTGTGCCCCTCCTGGCTAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCTTATTCTGTGCCCTGGGCATCCTGGCCACCTTCCACCTGCCCAAATGCTATGTACTTCTGTGGCTGCCAGAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAGCCCCAAGGAAGCATCAGATGGGAATAGTGGTAGTAGTGAGGCAACTCGGGGACACAGTGAATGA Rat T1R3 amino acid sequence--SEQ IDNO:25 MPGLAILGLSLAAFLELGMGSSLCLSQQFKAQGDYILGGLFPLGTTEEATLNQRTQPNGILCTRFSPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKPSLMFMAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELALITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGLSIFSGLANSRGICIAHEGLVPQHDTSGQQLGKVVDVLRQVNQSKVQVVVLFASARAVYSLFSYSILHDLSPKVWVASESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETRLALAADPTFCASLKAELDLEERVMGPRCSQCDYIMLQNLSSGLMQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHTSEPVQPWQLLENMYNMSFRARDLTLQFDAKGSVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQHSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTCTPCGKDQWSPEKSTTCLPRRPKFLAWGEPAVLSLLLLLCLVLGLTLAALGLFVHYWDSPLVQASGGSLFCFGLICLGLFCLSVLLFPGRPRSASCLAQQPMAHLPLTGCLSTLFLQAAEIFVESELPLSWANWLCSYLRGPWAWLVVLLATLVEAALCAWYLMAFPPEVVTDWQVLPTEVLEHCRMRSWVSLGLVHITNAVLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFIIWVSFVPLLANVQVAYQPAVQMGAILFCALGILATFHLPKCYVLLWLPELNTQEFFLGRSPKEASDGNSGSSEATRGHSE

1. A method of identifying a compound that modulates taste signaltransduction in taste cells, the method comprising the steps of: (i)contacting a test compound with an isolated taste receptor comprising atleast one T1R3 polypeptide and not comprising T1R1 or T1R2 and whereinthe T1R3 polypeptide is at least 90% identical to an amino acid sequenceof SEQ ID NO:15, 20, 23, 25 or 31; (ii) determining the functionaleffect of the test compound upon the receptor, wherein said functionaleffect is binding to the receptor or having an effect on the activity ofthe receptor; (iii) determining the functional effect of >300 mM naturalsugar on the receptor; and (iv) comparing the functional effect of thetest compound to the functional effect of >300 mM natural sugar and,identifying the test compound as the compound that modulates tastesignal transduction if the effect of the test compound is equal to orgreater than the effect of >300 mM natural sugar.
 2. The method of claim1, wherein the taste receptor comprises at least two T1R3 polypeptides.3. The method of claim 1, wherein the receptor is recombinant.
 4. Themethod of claim 1, wherein the receptor has G protein coupled receptoractivity.
 5. The method of claim 1, wherein the functional effect ismeasured in vitro.
 6. The method of claim 5, wherein the functionaleffect is a physical effect.
 7. The method of claim 5, wherein thereceptor is linked to a solid phase.
 8. The method of claim 5, whereinthe functional effect is determined by measuring binding of the compoundto the receptor.
 9. The method of claim 8, wherein the functional effectis determined by measuring binding of the compound to an extracellulardomain of the receptor.
 10. The method of claim 1, wherein the receptoris expressed in a cell or cell membrane, wherein the cell does notexpress T1R1 or T1R2.
 11. The method of claim 10, wherein the functionaleffect is a physical effect.
 12. The method of claim 11, wherein thefunctional effect is determined by measuring compound binding to thereceptor.
 13. The method of claim 12, wherein the functional effect isdetermined by measuring binding of the compound to an extracellulardomain of the receptor.
 14. The method of claim 10, wherein thefunctional effect is a chemical or phenotypic effect.
 15. The method ofclaim 14, wherein the functional effect is determined by measuringchanges in intracellular cAMP, IP3, or Ca2+.
 16. The method of claim 10,wherein the cell is a mammalian cell.
 17. The method of claim 16,wherein the cell is a human cell.
 18. The method of claim 16, whereinthe cell is a mouse cell.
 19. A method of identifying a modulator oftaste signal transduction in taste cells, the method comprising thesteps of (i) contacting a cell with a candidate compound and a naturalsweetener, wherein the cell expresses a taste receptor comprising a T1R3polypeptide and does not express T1R1 and T1R2; and (ii) determiningwhether the candidate compound modulates the functional effect of thenatural sweetener upon the receptor, thereby identifying the compound asthe modulator of taste signal transduction.